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https://f1000research.com/articles/4-26/v1	27 Jan 15	{ "type": "Research Note", "title": "Capsaicin from chili (Capsicum spp.) inhibits vascular smooth muscle cell proliferation", "authors": [ "Rongxia Liu", "Elke H. Heiss", "Dean Guo", "Verena M. Dirsch", "Atanas G. Atanasov", "Rongxia Liu", "Elke H. Heiss", "Dean Guo", "Verena M. Dirsch" ], "abstract": "Accelerated vascular smooth muscle cell (VSMC) proliferation is implied in cardiovascular disease and significantly contributes to vessel lumen reduction following surgical interventions such as percutaneous transluminal coronary angioplasty or bypass surgery. Therefore, identification and characterization of compounds and mechanisms able to counteract VSMC proliferation is of potential therapeutic relevance. This work reveals the anti-proliferative effect of the natural product capsaicin from Capsicum spp. by quantification of metabolic activity and DNA synthesis in activated VSMC. The observed in vitro activity profile of capsaicin warrants further research on its mechanism of action and potential for therapeutic application.", "keywords": [ "Capsaicin", "vascular smooth muscle cells", "restenosis", "proliferation" ], "content": "Main text\n\nAberrant and accelerated VSMC proliferation is a main contributor to restenosis, the pathological re-narrowing of the vessel lumen after surgical interventions combating vascular stenosis. To overcome restenosis, drug-eluting stents have been developed, aiming at inhibiting VSMC growth by the release of anti-proliferative substances such as paclitaxel and rapamycin. However, these compounds display unresolved issues such as impaired re-endothelialization and subsequent thrombosis induction1, which makes the characterization of other compounds able to suppress VSMC proliferation highly relevant. Plant-derived natural products are an excellent resource for identifying lead compounds2. Here we examine the anti-proliferative potential of capsaicin, a bioactive component of chili peppers [Capsicum spp. (Solanaceae)], in VSMC.\n\nTo test whether capsaicin is able to inhibit proliferation of VSMC induced by PDGF, a major growth factor implied in the aberrant proliferative responses in restenosis3, the total amount of metabolically active cells was measured after 48 h of incubation by the resazurin conversion method4. Capsaicin indeed suppressed VSMC proliferation concentration-dependently with an IC50 of 5.36 μM (Figure 1A). To confirm the anti-proliferative effect of capsaicin with a second experimental method, we measured DNA synthesis in VSMC by quantification of 5-bromo-2′-deoxyuridine (BrdU) incorporation into DNA. Capsaicin also inhibited PDGF-stimulated DNA synthesis in a concentration-dependent manner with an IC50 of 3.81 μM (Figure 1B). To assure that the decreased number of VSMC upon treatment with capsaicin is not due to cytotoxicity, we quantified cell death by measuring cell membrane integrity estimated by lactate dehydrogenase (LDH) activity inside cells and in cell supernatants. No significant cytotoxicity was detected in the investigated concentration range (Figure 1C). In summary, capsaicin is identified as an inhibitor of VSMC proliferation. Further studies are prompted to elaborate the underlying mode of action of this natural product and to investigate its effect in advanced in vivo anti-restenotic models.\n\nCell proliferation was estimated by quantification of metabolic activity (A) and DNA synthesis (B). Cell death was estimated by quantification of the percentage of extracellular LDH (C). Data represent mean ± SD from at least three independent experiments (n.s., not significant; *p < 0.001; p < 0.01; ANOVA/Bonferroni).\n\nRat aortic VSMC used in this study were purchased from Lonza (Braine-L’Alleud, Belgium) and cultivated in DMEM–F12 (1:1) medium supplemented with 20% fetal calf serum and gentamycin. Capsaicin and other chemicals were obtained from Sigma-Aldrich (Vienna, Austria).\n\nFor the resazurin conversion assay, VSMC were seeded in 96-well plates at 5 × 103 cells/well. 24 h later, cells were serum-starved for 24 h to render them quiescent. Quiescent cells were pretreated for 30 min with capsaicin or vehicle (0.1% DMSO) as indicated, and subsequently stimulated for 48 h with PDGF-BB (20 ng/mL). To measure the number of metabolically active VSMC by resazurin conversion4, cells were washed with PBS and incubated in serum-free medium containing 10 μg/mL resazurin for 2 h. Total metabolic activity was measured by monitoring the increase in fluorescence at a wavelength of 590 nm using an excitation wavelength of 535 nm in a 96-well plate reader (Tecan GENios Pro).\n\nFor the BrdU incorporation assay, VSMC were seeded and starved as for the resazurin conversion assay. Quiescent cells were pretreated for 30 min with capsaicin, or vehicle as indicated and subsequently stimulated with PDGF-BB (20 ng/mL). To estimate de novo DNA synthesis in VSMC5, BrdU was added 2 h after PDGF stimulation, and the incorporated amount was determined 22 h afterwards with a BrdU ELISA kit according to the manufacturer’s instructions (Roche Diagnostics).\n\nFor assessing cytotoxicity, VSMC were seeded and serum-starved as indicated above. The quiescent cells were pretreated for 30 min with capsaicin, or vehicle as indicated, and subsequently stimulated for 24 h with PDGF-BB (20 ng/mL). To quantify the loss of cell membrane integrity as a sign for cell death6, the supernatants of the treated cells were assessed for LDH activity. For estimation of the total LDH, identically treated samples were incubated for 45 min in the presence of 1% Triton X-100. The released and total LDH enzyme activity was quantified for 30 min in the dark in the presence of 4.5 mg/mL lactate, 0.56 mg/mL NAD+, 1.69 U/mL diaphorase, 0.004% (w/v) BSA, 0.15% (w/v) sucrose, and 0.5 mM 2-p-iodophenyl-3-nitrophenyl tetrazolium chloride (INT). The enzyme reaction was stopped with 1.78 mg/mL oxymate and the absorbance was measured at 490 nm in a 96-well plate reader (Tecan GENios Pro). Potential effects on cell viability were estimated as percentage of extracellular LDH activity. The cytotoxic natural product digitonin (100 μg/mL) was used as a positive control.\n\nStatistical analysis was performed by ANOVA/Bonferroni test (GraphPad PRISM software, version 4).\n\n\nData availability\n\nFigshare: Effect of capsaicin on vascular smooth muscle cell proliferation: raw data. doi: 10.6084/m9.figshare.12897217", "appendix": "Author contributions\n\n\n\nRL, DG, VMD and AGA conceived the study. RL, EHH and AGA designed and analyzed the experiments. RL performed the experiments. RL and AGA prepared the first draft of the manuscript. All authors critically commented on and revised the draft manuscript and agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the EU-FP7 Marie Curie Fellowship 252881, and by the University of Vienna “Back-to-Research Grant” (both to R. Liu); as well as by the Austrian Science Fund (FWF): S10704, P25971-B23, and P23317-B11.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nRaja SG: Drug-eluting stents and the future of coronary artery bypass surgery: facts and fiction. Ann Thorac Surg. 2006; 81(3): 1162–71. PubMed Abstract \| Publisher Full Text\n\nKinghorn AD, Pan L, Fletcher JN, et al.: The relevance of higher plants in lead compound discovery programs. J Nat Prod. 2011; 74(6): 1539–55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang XP, Pei ZH, Ren J: Making up or breaking up: the tortuous role of platelet-derived growth factor in vascular ageing. Clin Exp Pharmacol Physiol. 2009; 36(8): 739–47. PubMed Abstract \| Publisher Full Text\n\nKurin E, Atanasov AG, Donath O, et al.: Synergy study of the inhibitory potential of red wine polyphenols on vascular smooth muscle cell proliferation. Planta Med. 2012; 78(8): 772–8. PubMed Abstract \| Publisher Full Text\n\nSchwaiberger AV, Heiss EH, Cabaravdic M, et al.: Indirubin-3'-monoxime blocks vascular smooth muscle cell proliferation by inhibition of signal transducer and activator of transcription 3 signaling and reduces neointima formation in vivo. Arterioscler Thromb Vasc Biol. 2010; 30(12): 2475–81. PubMed Abstract \| Publisher Full Text\n\nFakhrudin N, Waltenberger B, Cabaravdic M, et al.: Identification of plumericin as a potent new inhibitor of the NF-κB pathway with anti-inflammatory activity in vitro and in vivo. Br J Pharmacol. 2014; 171(7): 1676–86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu R, Heiss EH, Guo D, et al.: Effect of capsaicin on vascular smooth muscle cell proliferation: raw data. Figshare. 2014. Data Source" }	[ { "id": "7483", "date": "30 Jan 2015", "name": "Carsten Gründemann", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the current manuscript, Rongxia Liu et al investigated the influence of the plant-derived natural product capsaicin to overcome restenosis by using a vascular muscle cell model(VSCM) in vitro.The manuscript is well written and conclusion is precise. The experiments are straightforward and the presented cell-based model is adequate to investigate such question. Nevertheless, before indexation the comments below have to be improved in a revised version of the manuscript.Major Comment:Main text: The authors mentioned that paclitaxel and rapamycin are used as standard drugs to inhibit VSCM proliferation. For pharmacological testing, it is essential that the authors should compare the investigated capsaicin to a standard therapy (positive control) to get an idea about capsaicin’s bio-activity.Minor Comment:Abstract: The authors have to depict more clearly why it is important to investigate new natural-derived compounds to overcome stenosis.", "responses": [ { "c_id": "1318", "date": "29 Apr 2015", "name": "Atanas Atanasov", "role": "Author Response", "response": "Thank you very much for taking the time to review our manuscript. While reporting a new bioactivity of the interesting natural product capsaicin, the purpose of our short note is not to claim superiority over existing VSMC inhibitors. Based on the literature, as well as on our own data, both suggested reference compounds, paclitaxel and rapamycin, are more potent than capsaicin. Nevertheless, the identification of new effective molecules (e.g., capsaicin) could be of potential interest, since it might serve as a starting point for the synthesis of more potent derivatives with superior bioactivity profiles (e.g., reduced adverse effects in vivo) in the future." } ] }, { "id": "7484", "date": "02 Feb 2015", "name": "Goutam Brahmachari", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have gone through the present manuscript entitled “Capsaicin from chili (Capsicum spp.) inhibits vascular smooth muscle cell proliferation” by Liu et al. with interest where the investigators demonstrated the efficacy of natural capsaicin in overcoming restenosis by using a vascular muscle cell model (VSMC) in vitro. Besides macrophages, vascular smooth muscle cells (VSMCs) are now also believed to play a significant role in generating foam cells that accumulate cytoplasmic droplets of cholesterol esters and triglycerides leading to atherosclerosis regarded as a prime cause of cardio- and cerebrovascular events. Proliferation of VSMCs thus is a common cause of restenosis among the patients who were previously undergone percutaneous transluminal coronary angioplasty or bypass surgery. Under this purview, the present work demonstrating promising in vitro anti-proliferative activity of natural capsaicin seems to be much interesting.The experiments are straightforward and sufficient in arriving at the conclusions. Representation of facts and organization of the manuscript are praise-worthy. The manuscript may be accepted and indexed in its present form. At the same time, I have a suggestion for the investigators as mentioned below, which may be addressed to make the revised manuscript more demanding.SuggestionRapamycin and paclitaxel are commonly used standard drugs to resist restenosis in patients although they suffer from certain serious health issues as mentioned by the authors in their present manuscript. Hence, search for better alternative(s) is warranted as a part of which the present work has been developed. Hence, I do suggest comparing the present results with those of at least any one of these two drugs (in addition to or in place of digitonin) to validate superiority of capsaicin.", "responses": [ { "c_id": "1319", "date": "29 Apr 2015", "name": "Atanas Atanasov", "role": "Author Response", "response": "Thank you very much for taking the time to review our manuscript. Based on the literature, as well as on our own data, the both suggested reference compounds, paclitaxel and rapamycin, are unfortunately more potent than capsaicin. Therefore with our short note we did not aim to claim superiority of action, but simply to report a new bioactivity of capsaicin, an interesting natural product with significant dietary and pharmacological relevance." } ] }, { "id": "7485", "date": "02 Feb 2015", "name": "Karel Šmejkal", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors are describing the inhibitory activity of capsaicin on vascular smooth muscle cell proliferation. The article looks to be well written, with adequate data. I miss only a bit deeper discussion of reasons for capsaicin selection for testing.", "responses": [ { "c_id": "1320", "date": "29 Apr 2015", "name": "Atanas Atanasov", "role": "Author Response", "response": "Thank you very much for taking the time to review our manuscript. Aiming to identify new inhibitors of VSMC proliferation, we tested a range of natural compounds derived from medicinal plants traditionally used for the treatment of different inflammation-associated conditions (including cardiovascular disease). One of the tested compounds was capsaicin, which revealed activity and therefore was further characterized." } ] } ]	1	https://f1000research.com/articles/4-26
https://f1000research.com/articles/4-25/v1	27 Jan 15	{ "type": "Research Note", "title": "Do Insurers Compete on the Federal Health Insurance Exchange?", "authors": [ "Jesse N. Cohen", "Alexander Coppock", "Arnab K. Ghosh", "Benjamin P. Geisler", "Alexander Coppock", "Arnab K. Ghosh", "Benjamin P. Geisler" ], "abstract": "Background: On the U.S. Federal Health Insurance Exchange established by the Affordable Care Act, states with fewer insurers have higher insurance premiums than states with more insurers. This expected feature of a competitive market has not been studied within states, however. We tested the hypothesis that insurance premiums decrease in more competitive geographic rating areas within states in the U.S.A.Methodology/principle findings: This cross-sectional study utilized publicly available premiums from the Federal Health Insurance Exchange website, www.healthcare.gov. Univariate and multivariate analyses were used to model premiums based on the number of insurers in geographic rating areas. The relationship between premiums and the number of insurers competing in a geographic rating area was also calculated for each unique insurance plan offered on the exchange. The data set and statistical code used for this research is linked in the publication. We found that there was an unexpected, marginally positive relationship between average monthly premiums and the number of insurers in a geographic rating area (+$5.71 in monthly premiums per additional insurer, p<0.001). We also found that identical plans tend to be offered with marginally higher premiums in rating areas with more insurers (+$3.18 in monthly premiums per additional insurer, p=0.002), contrary to the relationship we expected from a competitive marketplace. The principle limitation of the study is that this unexpected relationship, which suggests a lack of competitiveness of this early market, could be due to unobserved confounding factors that influence pricing in more competitive rating areas.Conclusion: On the Federal Health Insurance Exchange, the price of insurance is higher in more competitive rating areas within states. This may be explained by lack of competition in this early stage market.", "keywords": [ "Health", "Insurance", "Exchanges", "Patient", "Protection", "and", "Affordable", "Care", "Act", "Economic", "Competition" ], "content": "Introduction\n\nLack of competition in the U.S. health insurance market may contribute to higher prices for consumers, lower reimbursements for physicians, and greater profits for insurance companies1. In recent years, competition between insurers has been decreasing due to conglomeration of large insurers2. Through the Affordable Care Act (ACA), Congress attempted to improve the competitiveness of the insurance marketplace for individual buyers with the creation of online exchanges that facilitated comparison of plans and prices by consumers3. Nevertheless, consumer choice is still restricted by geography; not all plans are offered in all places. Most locations in the U.S. have few offerings, and identical plans are offered at different prices in different areas. As a result, many consumers still face a largely noncompetitive insurance market4–6.\n\nOn the exchanges established by the ACA, consumers must purchase plans from their own “rating areas”. The definition of a rating area changes considerably from state to state, but rating areas are typically a collection of similar counties within a state. They can also represent metropolitan statistical areas, and, in a few cases, the entire state. Prices for identical plans can vary across rating areas, but cannot vary within them. About half of U.S. counties on the exchanges belong to rating areas with only one or two insurers. Though states with fewer insurers have higher insurance prices on average7, this inverse relationship between price and the number of insurers per area has not been described at the level of within-state rating areas, which are the smallest geographic units of price variation on the federal health insurance exchange. Furthermore, no previous studies have tracked how insurers change the prices of identical plans that are offered in variably competitive rating areas. Since the negative correlation between the degree of competition and average state insurance prices could be due to confounding from interstate variations in cost of care, healthcare utilization8, or market share of healthcare provider organizations, it is worthwhile to investigate the relationship with finer granularity within states.\n\nThis article examines the hypotheses that average prices of insurance plans on the federal exchange decrease in more competitive rating areas within states, and that identical insurance plans are offered at lower prices in more competitive rating areas within states.\n\n\nMethods\n\nData were obtained from the Federal Health Insurance Exchange website, www.healthcare.gov, on October 17th 2013. The dataset contained all insurance plan offerings for 34 of the 36 states participating in the federal exchange. Idaho and New Mexico were not in the database. The operator of the federal exchange, the Centers for Medicare & Medicaid Services, listed premiums for different rating areas.\n\nWe defined a unique insurance plan as an offering by an insurance company with a unique plan name, metal tier (bronze, silver, etc.), network type (e.g., health maintenance organizations), and U.S. state. We excluded plans that had no monthly premiums listed. For precision of comparison, only family plans (two children and two forty year-old adults) were studied. Premiums for all other customer arrangements (single individuals for example) were related to the family plan premium based on a pre-set multiplier used by all insurers. The first analysis estimated the relationship between family plan premiums and the number of insurers per rating area using Ordinary Least Squares (OLS).\n\nThe second analysis estimated a varying-intercept, varying-slope model of family plan premiums and the number of insurers per rating area9. This multivariate linear regression model is equivalent to estimating separate regressions of price on the numbers of insurers per rating area for each unique insurance plan. This model is appropriate as there is considerable heterogeneity between plans in the relationship being studied (price on the number of insurers per rating area). The varying-intercept, varying-slope model effectively restricts the analysis to insurance plans that are offered in multiple rating areas with differing numbers of insurers, however. See Figure 1 and Figure 2 for a conceptual outline of the difference between our two models.\n\nPremium = β0 + β1 (Number of insurers). This model cannot capture the behavior of individual plans (A–C) in different rating areas, however.\n\nPremium = β0 + β1 (Number of insurers) + β2 (Insurance Plan A) + β3 (Insurance Plan B) + β4 (Insurance Plan ANumber of insurers) + β5 (Insurance Plan B Number of insurers). Where Insurance Plan A = 0,1 and Insurance Plan B = 0,1, and the base case is insurance plan C. Slope for Insurance Plan A, for example, is β1 + β4. Multiple regression lines are created with unique slopes and intercepts for each insurance plan. These slopes can be summarized in a histogram, as was done in our study.\n\nFigure 3 is a histogram of the slopes of the varying-intercept, varying-slope model, where each slope equals the change in premium associated with an increase of one insurer per rating area. The average slope was calculated and compared to zero using a t-test.\n\nAll statistical analyses were performed with R statistical package (R Project for Statistical Computing, www.r-project.org). The statistical code in R for our research is available at http://dx.doi.org/10.5281/zenodo.14122.\n\n\nResults\n\nThe Federal Health Insurance Exchange listed 2,276 unique plans offered by 139 insurers, in 34 states with 395 rating areas. The 2,276 unique plans were offered with different premiums based on the rating area. In total, there were 16,887 offerings across rating areas.\n\nThe number of rating areas per state varied from one (New Jersey, New Hampshire, and Delaware) to sixty-seven (Florida). See Figure 4 for the distribution of rating areas per state. The number of insurers per rating area varied from one to ten. 13% of rating areas had one insurer, 40% had two insurers (Figure 5).\n\nInsurance plans frequently changed prices across rating areas within a state. The average difference between the maximum and minimum prices, the range, for particular insurance plans across rating areas was $120. The largest range in the dataset was $1,459. Nearly all other plans ranged by less than $500 across rating areas, however.\n\nWe estimated the average relationship between family plan premiums and the number of insurers in a rating area by Ordinary Least Squares. The estimated slope was +$5.71 for each additional insurer (p<0.001). This simple model contradicts the hypothesized relationship in which increased competition decreased prices, but the model had an R2 of only 0.0007.\n\nTo more closely model how specific plans change prices in variably competitive rating areas, we estimated the relationship between competition and price for each unique plan separately. This was accomplished by a varying intercept, varying slope model, estimated by Ordinary Least Squares. We were able to estimate slopes for 1,512 of the 2,276 unique plans; the remainder were only offered in rating areas with equal numbers of insurers. The average slope for this model was +$3.18 for each additional insurer per rating area. This average could be distinguished from zero at p=0.002. The R2 of this more complicated model was 0.97, indicating much better model fit. The distribution of slopes from this model are presented in a histogram in Figure 3, which provides a sense of how varied the relationship between competition and price was.\n\n\nDiscussion\n\nOur hypotheses were that on the Federal Health Insurance Exchange, rating areas with more competitors would have lower average premiums than rating areas with fewer competitors, and that identical plans would be offered at lower prices in ratings areas with more competitors, presumably representing the effect of price competition. We found no evidence that increased numbers of insurers correlated with lower premiums, either for plans on average, or for plans tracked individually, however. We found instead that premiums increased marginally in rating areas with more competitors. This finding contradicts the previously documented negative correlation between premiums and the number of insurers as measured at the state level7.\n\nThere are multiple potential explanations for the contradiction of our hypothesis. Rating areas with more insurers may have higher medical or administrative costs, leading to higher premiums. Rating areas with more insurers may also be wealthier, leading to higher premiums through price discrimination. In contrast, if these areas are less wealthy, more consumers would be eligible for financial subsidization, possibly allowing higher prices to be offset by subsidies. In addition to the above factors, marginally higher prices in more competitive rating areas may simply reflect an absence of competition in this early market with an unknown customer base.\n\nEffects of competition in this online market may yet be seen as the “risk pool” becomes better understood, and pricing approaches market equilibrium. Alternatively, we may not see substantial price competition between insurers on this new market, as insurer price competition has only been documented in a small collection of studies, mostly limited to health maintenance organizations10.\n\nOur study had strengths in comparison to the previous literature. We examined the relationship between price and competition at the level of within-state rating areas, which yields a fine grained analysis compared to measuring the relationship between states. We may have reduced some geographic confounding using this method. We also tracked and summarized the pricing behavior of individual plans across areas with varying degrees of competition, which is a novel measurement of competitiveness.\n\nOur study was also subject to limitations. First, we did not capture the prices of plans that are offered off of the federal exchange, which is a potential underlying component of the competitive landscape in each rating area. Second, we reported on initial price offerings, not actual sales from 2014. Sales information would allow a calculation of the Herfindahl-Hirschman Index (HHI), an established metric of market competitiveness, but this data is not yet publically available. Lastly, we could not correct for confounding by rating area within states. Any model that examines premium variation as a factor of local competition will be left with the limitation that rating areas with higher numbers of insurers may simply be more expensive based on an unmeasured factor, which would confound a negative relationship expected in a competitive market. In short, there may be unobserved reasons that plans in rating area with more competition command higher premiums.\n\nIn conclusion, though an intention of the Federal Health Insurance Exchange was to foster competition between insurers and therefore lower insurance prices, we observed instead marginally higher prices in areas with more competition. This finding is important for policy makers, as the power of competition to lower health insurance prices is a theoretical underpinning of the Affordable Care Act.\n\n\nData availability\n\nF1000Research: Dataset 1. Healthcare.gov Insurance Exchange Prices for 2014, 10.5256/f1000research.6039.d4241311\n\nSource code in R: http://dx.doi.org/10.5281/zenodo.1412212", "appendix": "Author contributions\n\n\n\nJC, AG, BG conceived the study. JC, AC carried out all statistical analysis. JC was the primary author of the manuscript. JC, AC, AG, BG provided critical editing.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nWe acknowledge Mack Lipkin Jr., MD, and Kathleen Hanley, MD, for critical editing and invaluable mentorship.\n\n\nReferences\n\nAmerican Medical Association. Competition in health insurance: A comprehensive Study of U.S. Markets. 2007 Update. American Medical Association, 2007. Reference Source\n\nRobinson JC: Consolidation and the transformation of competition in health insurance. Health Aff (Millwood). 2004; 23(6): 11–24. PubMed Abstract \| Publisher Full Text\n\nSkopec L, Kronick R: Market Competition Works: Silver Premiums in the 2014 Individual Market Are Substantially Lower than Expected. Office of the Assistant Secretary for Planning and Evaluation. Department of Health and Human Services. 2013. Reference Source\n\nAbelson R, Thomas K, McGinthy JC: Health Care Law Fails to Lower Prices For Rural Areas. New York Times. 2013. Reference Source\n\nSenger A: Lack of Competition in Obamacare’s Exchanges: Over Half of U.S. Has Two or Fewer Carriers. The Heritage Foundation. Retrieved June 4, 2014. Reference Source\n\nCox CM, Rosa CG, Levitt L: Sizing Up Exchange Market Competition. The Henry J. Kaiser Family Foundation. Issue Brief. 2014. Reference Source\n\nHealth Insurance Marketplace Premiums for 2014. Office of the Assistant Secretary for Planning and Evaluation. Department of Health and Human Services. 2013; 4. Reference Source\n\nGottlieb DJ, Zhou W, Song Y, et al.: Prices don’t drive regional Medicare spending variations. Health Aff (Millwood). 2010; 29(3); 537–43. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGelman A, Jennifer H: Data Analysis Using Regression and Multilevel/Hierarchical Models. 2007. Reference Source\n\nDicken JE: Private Health Insurance: Research on Competition in the Insurance Industry. United States Government Accountability Office. 2010; 1–13. Reference Source\n\nCohen JN, Coppock A, Ghosh A, et al.: Dataset 1. Healthcare.gov Insurance Exchange Prices for 2014. F1000Research. 2015. Data Source\n\nCohen JN, Coppock A, Ghosh A, et al.: Replication Code in R for “Do Insurers Compete on the Federal Heath Insurance Exchange. Zenodo. 2015. Data Source" }	[ { "id": "7477", "date": "16 Feb 2015", "name": "Cynthia Cox", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study analyzes the relationship between Federally Facilitated Marketplace (FFM) premiums and the number of insurers participating in the region. The authors present a good overview of the dataset used, existing literature, and limitations in the availability of data (particularly the lack of plan-level signup data, which would allow for examination of market concentration).The use of family premiums is appropriate, but as the authors note, age curves are the same in almost all FFE states, so single adult premiums would have yielded similar results (though the dollar amounts would be smaller). Rating regions are also the appropriate level of analysis. The analysis could be repeated with the second year of data (2015) as this has been publicly available for some time.The study consists of two parts: 1) simple regression where average premium is the dependent variable and 2) a varying-intercept, varying-slope model of the relationship between unique plans’ premiums and the number of insurers in the rating area. As I will describe in more detail below, the first analysis has significant flaws/limitations. I would suggest minor revisions and elaboration on the second analysis.On the first analysis, my concern is that the dependent variable should not be the average premium in the rating area. Rather, the dependent variable should be the lowest and/or second-lowest cost premium by metal level in the rating area. This is for two reasons:First, conceptually, exchange subsidies are structured such that insurers have an incentive to compete to offer the lowest-cost product within each metal level (or possibly the benchmark in the case of silver plans). We saw from 2014 HHS enrollment reports that the majority of consumers purchased one of the two lowest cost silver or bronze plans.Second, in urban areas, where more insurers participate, a wider range of products are typically offered within each metal level, and insurers may also be more likely to offer higher-cost platinum coverage. A wide range of products may result in higher average premiums, even while insurers are competing to offer the lowest-cost product within a metal level.The second analysis has potential to make a contribution to the literature and bears elaboration. One small methodological issue, though, is the use of plan names, rather than the plan ID standard component, to identify unique plans. Insurers may offer similar plans that are listed with the same marketing name in the dataset but in reality have slight variations. In some cases this is because one plan may have a narrower network (and therefore lower premium), and in other cases it may be that the insurer filled out the plan marketing name incompletely.For example, FirstCare Health Plans in San Saba County Texas offers two gold HMO plans with marketing names of “FirstCare Health Plans” but the plan IDs differ (26539TX0140001 and 26539TX0140002), and they have slightly different premiums ($310.39 and $305.89, respectively, for a 40-year-old individual). From the dataset alone, we do not know whether this is simply an error in the marketing name or that the two plans are similar other than their networks, but in either case, they should be counted as two distinct plans. An alternative would be to use a combination of unique plan marketing names and unique premiums within the rating area to identify unique plans. To be clear, I doubt the use of plan IDs vs. plan names to identify unique plans would have a significant effect on the overall findings, but it would be a cleaner analysis.Finally, I would like to see more elaboration on the second analysis. For example, I am interested in hearing more about the plans that are only offered in one rating area or that did not vary in price between rating areas. Are these plans lower cost, on average, relative to the insurers’ other products in those areas? More elaboration on the excluded plans would be beneficial.", "responses": [] }, { "id": "8034", "date": "19 Mar 2015", "name": "Jon Gabel", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWhen Results Seem Implausible, It Is Wise to Take a Second LookJesse Cohen and his colleagues present their analysis of the relationship between premiums on the exchange and the number of insurers in a market area, and conclude that premiums rise $5.71 for each additional insurer on the exchange. Their analysis is based on data from 34 states on the Federally Facilitated Marketplaces (FFMs) in 2014. Cohen and his colleagues conclude that in the early stages, there is a lack of price competition on the exchanges among insurers.There is a lengthy history in the health economics profession debating counter-intuitive findings. In the late 1970s and 1980s, many economists, including myself, pointed to the higher physician fees in areas with more physicians per capita, and argued this was consistent with physician-induced demand. Similarly, during the 70s and 80s, there was wide acceptance of the proposition that if a hospital could add additional beds, the beds would be filled. But times have changed, and the fee-for-service and indemnity insurance world of the 70s and 80s is long gone. Activist employers and managed care have helped put many of the older controversies to rest, and a strategy of competition is now embraced by both parties. To build a price competitive Marketplaces, the Affordable Care Act borrowed from the playbook of conservative economists such as Alain Enthoven and Mark Pauly1,2. Subsidies are pegged to the second lowest silver plan, so that the cost of a higher cost plan is borne entirely by the purchasing household. Essential benefits, metal tiers based on actuarial values and transparency offered through state websites promote price sensitive buying. Rather than illustrating a lack of competition among insurers, current evidence suggests that the exchanges are bringing price competition into the non-group market not witnessed in many years. Premiums cost 16 percent less than those predicted by the Congressional Office in 20143. Sixty-four percent of 2014 Marketplace enrollment was for the lowest or second lowest cost plan on the metal tier in the rating area4. There was a 25 percent increase in the number of carriers in 2015 competing on the Marketplaces and in some states new entrants gained not just a plurality of enrollment, but a majority5. The number of plans offered rose by 25 percent. Some new entrants gaining the largest market share in 2014 went bankrupt or exited the market in 2014-20156. This occurred in Iowa, Nebraska, and Minnesota for two co-op plans and a hospital-based plan. Between 2014 and 2015 the average cost of Marketplace plans did not increase. Deductibles also remained essentially unchanged. Pricing of plans followed a regression toward the mean pattern, with low cost 2014 plans increasing and high cost plans reducing their premiums. So connecting the dots, it appears the Marketplaces were built to be and are price competitive5.Analysis of Cohen and ColleaguesThe authors conducted two sets of analyses. In the first, using ordinary least squares regression, they estimated the relationship between the number of insurers in a rating area and the average premium. In the second, they use a variable slope, variable intercept model which the authors describe as equivalent to estimate separate regression for each plan offered in multiple areas within a state. It is from these second analyses that the authors reach their conclusion that as the number of insurers within a market area increases, premiums likewise increase. I thought it was insightful that the authors examined premiums for the same plan in different market areas within a state.Cohen and colleagues themselves point out that there may be intervening variables that explain these unexpected findings – that areas with more insurers have higher premiums. Large states have more insurers participating on the exchanges and small rural states may have but one insurer participating. Within a state, rural areas tend to have fewer insurers participating compared to urban and suburban areas. Medical expenses, particularly hospital expenses, are higher in urban and suburban areas. Physician practice expenses such as rent and labor are greater in urban/suburban areas. However, in some rural areas local hospitals have de facto monopolies, and in some of these areas such as southern rural Georgia, premiums are very high.Before we accept the authors’ conclusions, I suggest that they control for the many intervening variables. These include; (1) area per capita income (2) some measure of average hospital costs in the rating area (3) percent of the population that is uninsured in the rating area (4) some measure of “urbanness” (5) type of insurer (Blue Cross Blue Shield, major commercial carrier, co-op, provider-based, Medicaid base etc. One other concern I have is that certain states may have undue weight. For example, in Florida, Governor Rick Scott and the Republican controlled legislature, to thwart the implementation of the ACA, divided the state into 58 regions. So Florida would have undue influence on findings. I suggest weighting each area by the uninsured population or total population.In fairness to Cohen and his colleagues, the authors note that the results may reflect the early stages of competition on the Marketplaces. Now that 2015 data are available for FFS states, I invite the authors to pool two years of data and determine if there initial observations still hold, with a fully specified model.", "responses": [] } ]	1	https://f1000research.com/articles/4-25
https://f1000research.com/articles/3-251/v1	24 Oct 14	{ "type": "Research Note", "title": "Characterizing alpha helical properties of Ebola viral proteins as potential targets for inhibition of alpha-helix mediated protein-protein interactions", "authors": [ "Sandeep Chakraborty", "Basuthkar J. Rao", "Bjarni Asgeirsson", "Abhaya M. Dandekar", "Basuthkar J. Rao", "Bjarni Asgeirsson", "Abhaya M. Dandekar" ], "abstract": "Ebola, considered till recently as a rare and endemic disease, has dramatically transformed into a potentially global humanitarian crisis. The genome of Ebola, a member of the Filoviridae family, encodes seven proteins. Based on the recently implemented software (PAGAL) for analyzing the hydrophobicity and amphipathicity properties of alpha helices (AH) in proteins, we characterize the helices in the Ebola proteome. We demonstrate that AHs with characteristically unique features are involved in critical interactions with the host proteins. For example, the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain has an AH with a large hydrophobic moment. The ability of this AH to bind to other host proteins is disrupted by a neutralizing antibody derived from a human survivor of the 1995 Kikwit outbreak, emphasizing the critical nature of this secondary structure in the virulence of the Ebola virus. Our method ensures a comprehensive list of such `hotspots'. These helices probably are or can be the target of molecules designed to inhibit AH mediated protein-protein interactions. Further, by comparing the AHs in proteins of the related Marburg viruses, we are able to elicit subtle changes in the proteins that might render them ineffective to previously successful drugs. Such differences are difficult to identify by a simple sequence or structural alignment. Thus, analyzing AHs in the small Ebola proteome can aid rational design aimed at countering the `largest Ebola epidemic, affecting multiple countries in West Africa' (http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/index.html).", "keywords": [ "The Ebola virus was first discovered in 19761", "and has been since known as a rare", "but deadly disease2. However", "the current outbreak in West African countries (Guinea", "Liberia", "Nigeria", "Sierra Leone and Senegal) has rapidly deteriorated into a full blown epidemic3", "and poses grave humanitarian dangers to these countries4. Ebola", "along with the Marburg virus", "belongs to the Filoviridae family5", "and causes haemorrhagic fever2 by quickly suppressing innate antiviral immune responses to facilitate uncontrolled viral replication6." ], "content": "Introduction\n\nThe Ebola virus was first discovered in 19761, and has been since known as a rare, but deadly disease2. However, the current outbreak in West African countries (Guinea, Liberia, Nigeria, Sierra Leone and Senegal) has rapidly deteriorated into a full blown epidemic3, and poses grave humanitarian dangers to these countries4. Ebola, along with the Marburg virus, belongs to the Filoviridae family5, and causes haemorrhagic fever2 by quickly suppressing innate antiviral immune responses to facilitate uncontrolled viral replication6.\n\nInterestingly, the genome of the Ebola virus encodes seven proteins7, although their extreme ‘plasticity allows multiple functions’8,9. Protein structures are formed by well ordered local segments, of which the most prevalent are alpha helices (AH) and β sheets. AHs are right-handed spiral conformations which have a hydrogen bond between the carbonyl oxygen (C=O) of each residue and the alpha-amino nitrogen (N-H) of the fourth residue away from the N-terminal. AH domains are often the target of peptides designed to inhibit viral infections10–12. Recently, we have provided open access to software that has reproduced previously described computational methods13 to compute the hydrophobic moment of AHs (PAGAL14).\n\nIn the current work, we characterize the helices in the Ebola proteome using PAGAL, and demonstrate that the helices with characteristically unique feature values are involved in critical interactions with the host proteins. The PDB database is queried for the keyword ‘Ebola’, and the structures obtained are analyzed using DSSP (Define Secondary Structure of Proteins)15 for identifying AHs. We process all PDB structures, and do not filter out redundant structures based on sequence. These helices are analyzed using PAGAL, and the results are sorted based on three criteria - hydrophobic moment and high proportion of positive or negative residues. The helices that are ranked highest in these sorting criteria are involved in critical interactions with either antibodies or host proteins. For example, the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain has an AH with the largest hydrophobic moment in all helices analyzed16. This helix is disrupted by a neutralizing antibody derived from a human survivor of the 1995 Kikwit outbreak, emphasizing the critical nature of this helix in the virulence of Ebola17. Another example, obtained by choosing the helix with the highest proportion of negatively charged residues, is the interaction between the human karyopherin alpha nuclear transporters C terminus and the Ebola virus VP24 protein (eVP24)18, which suppresses tyrosine-phosphorylated STAT1 nuclear import19. These helices probably are, or can be, the target of molecules designed to inhibit AH mediated protein-protein interactions20. Our method provides a comprehensive list of such targets. Further, each protein can be individually queried using PAGAL, and thus identified helices that might have a poor global rank, but still be critical in the particular proteins context.\n\nAlthough, Ebola and Marburg viruses are members of the Filoviridae family21, they have different antigenicity of the virion glycoprotein22. These differences are probably the rationale for the lesser mortality observed in Marburg outbreaks. By comparing the AHs in proteins of Marburg and Ebola viruses, we are able to elicit subtle changes in the proteins that might render them ineffective against previously successful drugs. These differences are not apparent from a simple sequence or structural alignment. Thus, in the current work, we elucidate a simple methodology that can aid rational design of drugs and vaccine, an important aspect of the global effort to counter the deadly Ebola epidemic.\n\n\nMaterials and methods\n\nWe searched for the keyword ‘Ebola’ in the PDB database (Table 1). Subsequently, each protein was split based on the chain ID, resulting in 146 single chained proteins (See ALPHA.zip in Dataset 1). We have not reduced the set based on sequence similarity since the proteins might have different conformations based on their ligands. Note, this list might include non-Ebola proteins which might have been co-crystallized with the Ebola protein. However, they have been put through the same analysis since they might provide insights into the Ebola proteins themselves.\n\nThese proteins were then analyzed using DSSP15, and resulted in 758 helices in all (See ALPHA.zip in Dataset 1). These helices were then analyzed using PAGAL. The PAGAL algorithm has been detailed previously14. Briefly, the Edmundson wheel is computed by considering a wheel with centre (0,0), radius 5, first residue coordinate (0,5) and advancing each subsequent residue by 100 degrees on the circle, as 3.6 turns of the helix makes one full circle. We compute the hydrophobic moment by connecting the center to the coordinate of the residue and give it a magnitude obtained from the hydrophobic scale (in our case, this scale is obtained from13). These vectors are then added to obtain the final hydrophobic moment.\n\nThe color coding is as follows: all hydrophobic residues are colored red, while hydrophilic residues are colored in blue: dark blue for positively charged residues, medium blue for negatively charged residues and light blue for amides.\n\nThe raw file generated by analyzing all 146 proteins through PAGAL is provided as PAGALRAW-DATA.txt (Dataset 1), and contains the hydrophobic moment, percent of positive charges and the total number of charged residues for every helix. These are then sorted based on the charge (negative or positive) or the hydrophobic moment. We ignore the helices that have none or a single one charged residue.\n\nAll protein structures were rendered by PyMol (http://www.pymol.org/). The sequence alignment was done using ClustalW23. The alignment images were generated using Seaview24. Protein structures have been superimposed using MUSTANG25.\n\n\nResults and discussion\n\nWe began by analyzing the helices which have a large hydrophobic moment (hydrophobic scale is obtained from13) (Table 2). The Edmundson wheel for the helix 1EBOE.HELIX1 from the structure of GP2 from the Ebola virus membrane fusion glycoprotein (PDBid:1EBO)16 is shown in Figure 1a. Figure 1b shows the residues comprising these helices (in magenta) in the apo form (PDBid:1EBO)16. This helix is disrupted by a neutralizing antibody derived from a human survivor of the 1995 Kikwit outbreak (PDBid:3CSY)17, emphasizing the critical nature of this helix in the virulence of Ebola (Figure 1c,d). Table 3 shows the residues in the specified helix (residues 553–597, chain J, PDBid:3CSY) making possible hydrogen bonds with different residues in the human Fab KZ52 heavy chain (residues 1–228, chain A, PDBid:3CSY). Among all the interactions, only Gly553 is on 1EBOE.HELIX1 (at a distance of 2.7 Å from Thr100/OG1), although the others are sequentially proximal. These few interactions are sufficient to disrupt this helix, rendering the virus non-virulent, and leading to human recovery. The importance of interfacial hydrophobicity in viral proteins involved in host entry through membrane fusion has recently been discussed in details, and remains ‘an underutilized therapeutic target’26. It is also interesting that the helix is also involved in a disulphide bond after its disruption (Cys556 and Cys511). 1EBOE.HELIX0 (Table 2) also has a high hydrophobic moment, but is actually an isoleucine zipper derived from GCN427 (Figure 1b).\n\nProperty based on which the sorting is done is either the Hydrophobic moment (HM) and the percentage of negative (NEG) or positive residues (POS). HM: Hydrophobic moment, RPNR: Ratio of the positive to the negative residues, Len: length of the helix, NCH: number of charged residues, GP: glycoprotein from Ebola, VP24: Membrane-associated protein from Ebola, VP35: Polymerase cofactor.\n\n(a) Edmundson wheel for 1EBOE.HELIX1. The hydrophobic moment vector is not to scale. The color coding is as follows: all hydrophobic residues are colored red, while hydrophilic residues are colored in blue: dark blue for positively charged residues, medium blue for negatively charged residues and light blue for amides. (b) Structure of PDBid:1EBOE, 1EBOE.HELIX1 is marked in magenta and the leucine zipper is in blue. (c) 1EBOE.HELIX1 is disrupted by an antibody derived from a human survivor of the 1995 Kikwit outbreak (PDBid:3CSY). (d) Gly553/N on 1EBOE.HELIX1 makes a possible hydrogen bond to Thr100/OG1 at a distance of 2.7 Å.\n\nThe helix with a large hydrophobic moment, as determined from PDBid:1EBOE, is disrupted in the structure from PDBid:3CSY through possible hydrogen bonds with different residues in the human Fab KZ52 heavy chain (antibody, chain A). The helix residues are: 553-597 in chain J, PDBid:3CSY.\n\nWe then analyzed the helices having a high proportion of negatively charged residues, sorted based on the length of the helix when the percentage of negatively residues are the same (Table 2). Figure 2a shows the Edmundson wheel for the helix 4U2XA.HELIX5 (which has only two charged residues - the basic E113 and D124), while Figure 2b,c shows this helix in the protein complex marked in magenta. Protein PDBid:4U2XD is the human karyopherin alpha nuclear transporter (KPNA) C terminus in complex with the Ebola virus VP24 protein (eVP24)18. eVP24 interferes with the immune response by selectively targeting tyrosine-phosphorylated STAT1 nuclear import19. It does not hinder the transport of other cargo that may be required for viral replication. 4U2XA.HELIX5 is responsible for forming the complex with the KPNA protein through a helix (4U2XD.HELIX9, in blue), and K481 from KPNA is in contact with D124 from eVP24 (distance between K481/NZ and D124/OD2 is 3.98 Å). Their interaction is probably electrostatic, since the atoms have opposite charges. VP24 has also been shown to directly bind to STAT1, further compromising the immune response28.\n\n(a) Edmundson wheel for 4U2XA.HELIX5. (b) Complex of VP24 (PDBid:4U2XA) and human karyopherin alpha nuclear transporters (KPNA) C terminus (PDBid:4U2XD). (c) D124 from VP24 probably has an electrostatic interaction with K481 from KPNA. This interaction is sufficient to interfere with the immune response to Ebola infection.\n\nThe next helix having a high proportion of negatively charged residues (3FKEA.HELIX2) is from a VP35, a classic example of a moonlighting protein, that can be a component of the viral RNA polymerase complex, a viral assembly factor, or inhibitor of host interferon production29. We have not been able to identify a critical role for this helix in the protein from current literature. However, VP35 consists of several helices, and is reasonably conserved in the Marburg virus from the same Filoviridae family (42% identity, 58% similarity) (Figure 3a). Often, it is difficult to identify the regions of the protein that differ from a sequence or structural alignment (Figure 3b), in case one is interested in understanding different responses of the proteins to known drugs or even the immune system. Table 4 compares the characteristics of the helices in the VP35 from Ebola and Marburg (the helix numbering is offset by one, due to a small N-terminal helix in the Marburg protein (which might be due to crystallization technique differences and probably is not critical). Thus, we have numbered these helices using alphabets. It can be seen that most of the helices have the same properties, barring helices E and F, where the acidic residue is present in the E helix in Marburg and in the F helix in Ebola. These helices are marked in yellow in Figure 3b. Also, it can be seen that helix C, which has a high proportion of acidic residues in VP35, has a fewer number of those residues in Marburg. Marburg outbreaks (http://www.who.int/mediacentre/factsheets/fs_marburg/en/) have been fewer in comparison to Ebola outbreaks (http://www.who.int/mediacentre/factsheets/fs103/en/). It is known that even for Ebola, the Zaire strain had a much higher mortality rate than the Sudan one30. These differences are definitely encoded in the proteins expressed by these viruses, and the design of drugs and vaccines to counter them should take these differences into account.\n\nVP35 has several moonlighting functions related to immune evasion. (a) Sequence alignment of VP35 from Marburg (PDBid:4GHLA) and Ebola (PDBid:3FKEA). (b) Structural alignment using MUSTANG. The helices that have differing properties are marked in yellow. 3FKEA.HELIX1 spanning residues 238–252 is marked in magenta. This is a helix with a high proportion of positively charged residues that have been observed to have important interactions in the structure29. (c) Edmundson wheel for 3FKEA.HELIX1.\n\nComparing the VP35 protein from Marburg (PDBid:4GHLA) and Ebola (PDBid:3FKEA). Note the helices are offset by one, due the presence of an extra helix in the Marburg VP35. Thus, we name the helices using alphabets. It can be seen that most helices have the same properties, barring helices E and F, where the acidic residue is present in the E helix in Marburg and in the F helix in Ebola. HM: Hydrophobic moment, RPNR: Ratio of the positive to the negative residues, Len: length of the helix, NCH: number of charged residues.\n\nFor helices having a high proportion of positively charged residues, we could not find any reference to the critical nature of the first helix (Table 2, 4U2XA.HELIX7). This helix is marked in yellow in Figure 2c. The second helix (3FKEA.HELIX1) is from VP35, which was discussed previously29. This helix spans residues 238–252 and includes Lys248 and Lys251, a basic patch which is ‘100% identical among members of the Ebola viral isolates’29, and Ala238, Gln241, Leu242, Val245, Ile246, Leu249 which interacts with a β sheet to create a hydrophobic subdomain29. This helix is marked in magenta in Figure 3b, and the Edmundson wheel is shown in Figure 3c. Once again, we demonstrate that unique values of an AH is a strong indicator of its significance in the viral functionality.\n\nThe multifunctional roles played by many of these Ebola proteins is probably due to stretches of intrinsically disordered regions within the structure - ‘fuzzy objects with fuzzy structures and fuzzy functions’31. The conformational plasticity9 and moonlighting abilities of these proteins are key determinants for immune evasion32.\n\nThe above examples have analyzed all helices from the Ebola proteome. However, it also possible to analyze the helices in a single protein, and probe those for unique features. Table 5 shows the values obtained from PAGAL for helices of the C-terminal domain of the Zaire Ebola virus nucleoprotein33. It can be seen that 4QAZA.HELIX0 (residues 646–658) has a reasonably high hydrophobic moment (although it will not rank highly if we analyze all helices from the proteome), and also a high number of charged residues (Figure 4a,b). It has been observed that ‘the side chains of Glu645, His646, Glu649, Lys684, Glu695, Glu709, Lys728 and Gln739 are partly disordered so that some or all of their atoms are not visible in the electron density’33. Glu645, His646, Glu649 are part of this helix, and are thus critical to the disorderedness of the protein, which is critical for its moonlighting roles. Note, that Glu has been observed to be the second most disorder promoting residue (after proline)34. Furthermore, Tyr652 and Leu656, which lie in this helix, are residues that have been hypothesized to be part of the protein-protein interaction site involving this protein33.\n\n4QAZA.HELIX0 comprising of residues 646-658 has a reasonably large hydrophobic moment, and has been hypothesized to be part of the protein which is involved in protein-protein interactions33. Further, these helices have residues with disordered sidechains33, which are known to be critical for moonlighting functions31. HM: Hydrophobic moment, RPNR: Ratio of the positive to the negative residues, Len: length of the helix, NCH: number of charged residues.\n\n(a) Edmundson wheel for 4QAZA.HELIX0 (residues 646-658). (b) Protein structure for PDBid:4QAZA.\n\n\nConclusions\n\nThe ability of a genome as small as the Ebola virus to inflict a dishearteningly high percentage of mortality in human subjects is a humbling experience in the context of the tremendous technological advancements achieved in the last few decades3,4. The Ebola virus potently suppresses the human immune response2,6,35 by binding with key human proteins involved in the immune pathway18. These protein-protein interactions are often mediated through well structured secondary regions within the protein structures (alpha helices), and the design of molecules that inhibit these ‘hotspots’20,36 has been a well known strategy to develop drugs to counter bacterial and viral infections10–12. For example, synthetic peptides derived from the oligomerization domain of polymerase subunits has been shown to inhibit viral proteins37,38. On the other hand, there might exist other protein domains that might be exploited by non-native viral peptides to obstruct viral functionality. In the current work, we characterize alpha helices in the Ebola virus proteome using a recently implemented open access software (PAGAL)14, thus identifying potential targets for inhibition of the helix mediated interactions. Through several examples, we demonstrate that helices with unique features are involved in interactions with host proteins (either antibodies from survivors, or proteins regulating the immune response). Further, we also provide an alternate way of analyzing differences in related proteins (from the Marburg virus) by focusing on the properties of corresponding helices. As future work, we intend to develop methodologies to design peptides that would target these ‘hotspots’36. It has to be kept in mind that it has been a challenge to design small ligands that disrupt protein-protein interactions, and designers resort to several innovative techniques to overcome thermodynamic instability or proteolytic susceptibility39–42. These helices can essentially be epitopes43,44 for developing antibodies against the virus45,46. Interestingly, ZMapp, a cocktail of three antibodies has shown reversion of advanced Ebola symptoms in non-human primates47, and uses only glycoprotein-specific epitope generated antibodies44,48. It is interesting to hypothesize that additions to this cocktail with antibodies derived from other epitopes (for example, 4U2XA.HELIX5 from VP24 that is involved in immune response suppression) could prove more effective. Thus, we provide a comprehensive list of potential targets from the small proteome of the Ebola virus that can directed rational design to quickly innovate therapies.\n\n\nData availability\n\nF1000Research: Dataset 1. PAGAL analysis of Ebola-related alpha helices, 10.5256/f1000research.5573.d3745349", "appendix": "Author contributions\n\n\n\nSC wrote the computer programs. All authors analyzed the data, and contributed equally to the writing and subsequent refinement of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nAMD wishes to acknowledge grant support from the California Department of Food and Agriculture PD/GWSS Board. BJ acknowledges financial support from Tata Institute of Fundamental Research (Department of Atomic Energy). Additionally, BJR is thankful to the Department of Science and Technology for the JC Bose Award Grant. BA acknowledges financial support from the Science Institute of the University of Iceland.\n\n\nReferences\n\nPattyn S, van der Groen G, Courteille G, et al.: Isolation of Marburg-like virus from a case of haemorrhagic fever in Zaire. Lancet. 1977; 1(8011): 573–574. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTakada A, Feldmann H, Stroeher U, et al.: Identification of protective epitopes on ebola virus glycoprotein at the single amino acid level by using recombinant vesicular stomatitis viruses. J Virol. 2003; 77(2): 1069–1074. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilson JA, Hevey M, Bakken R, et al.: Epitopes involved in antibody-mediated protection from Ebola virus. Science. 2000; 287(5458): 1664–1666. PubMed Abstract \| Publisher Full Text\n\nTakada A, Ebihara H, Jones S, et al.: Protective efficacy of neutralizing antibodies against Ebola Virus infection. Vaccine. 2007; 25(6): 993–999. PubMed Abstract \| Publisher Full Text\n\nQiu X, Alimonti JB, Melito PL, et al.: Characterization of Zaire ebolavirus. glycoprotein-specific monoclonal antibodies. Clin Immunol. 2011; 141(2): 218–227. 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Data Source" }	[ { "id": "6506", "date": "04 Nov 2014", "name": "Winfried Weissenhorn", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors suggest that they can identify alpha helices and predict their propensities to be targeted by small molecules. Their test case is the small Ebola virus genome, where several crystal structures are available.First they compute the hydrophobic moment of identified helices with their previously published program PAGAL and classify them based on hydrophobicity, positive or negative charges. They conclude that helices with unique feature values are involved in host protein interaction.Page 4: It is not correct to state that “ this helix is disrupted by a neutralizing antibody derived from a human survivor …”. HR1 or helix 1 from Gp2 is split into 4 small helices in the native GP structure and antibody binding prevents its refolding into the post fusion conformation represented by the Gp2 structure. Now one can argue that small molecules could interfere with the formation of the triple stranded coiled coil formed by HR1 in the post fusion structure. This needs to be clarified in the text.Next they identified a charged helix in Vps24 that interacts with karyopherin. Why was this chosen? Because of the available structure? This helix contains only two charged residues and would not fall under the classification of carrying a high charge!The third helices described in detail are from Vps35 and the authors identify several helices with carry charges, but no clear targets are discussed.Page 6: The authors make a connection between the number of acidic residues in a helix from Ebola Vps35 compared to Marburg Vps35 and the frequency of outbreaks, which is a complete over interpretation of their data.In summary the manuscript describes an interesting approach to identify or validate potential drug targets. However, the authors need to be more cautious in interpreting their results. Without any experimental validation their approach to link helical properties to protein interaction propensities is extremely weak.", "responses": [ { "c_id": "1067", "date": "06 Nov 2014", "name": "Sandeep Chakraborty", "role": "Author Response", "response": "Dear Dr Weissenhorn,We would like to thank you for taking the time to review this paper, and for your suggestions to improve the manuscript. In the interim period, we have applied other computational methods1 to correlate the different immunosuppressive and pathogenicity mechanisms in Ebola and Marburg viruses to variations in their structures/sequences 2. Please find our detailed responses to yourcomments below.The authors suggest that they can identify alpha helices and predict their propensities to be targeted by small molecules. Their test case is the small Ebola virus genome, where several crystal structures are available.First they compute the hydrophobic moment of identified helices with their previously published program PAGAL and classify them based on hydrophobicity, positive or negative charges. They conclude that helices with unique feature values are involved in host protein interaction.Page 4: It is not correct to state that this helix is disrupted by a neutralizing anti- body derived from a human survivor . HR1 or helix 1 from Gp2 is split into 4 small helices in the native GP structure and antibody binding prevents its refolding into the post fusion conformation represented by the Gp2 structure. Now one can argue that small molecules could interfere with the formation of the triple stranded coiled coil formed by HR1 in the post fusion structure. This needs to be clarified in the text.We appreciate this point, (‘KZ52 likely neutralizes by preventing rearrangement of the GP2 HR1A/HR1B segments and blocking host membrane insertion of the internal fusion loop’ 3), and have made the correction.Next they identified a charged helix in Vps24 that interacts with karyopherin. Why was this chosen? Because of the available structure? This helix contains only two charged residues and would not fall under the classification of carrying a high charge!VP24 came up in the sorted list since it has a ‘high proportion of negatively charged residues’, and not high charge. The proportion of charged residues is computed based on the total number of charged residues, and not the length of the helix. We could also create a category of high charge by combining the previous feature (high proportion) to high number of charged residues.Our search criteria excludes AHs with zero or one charged residue. We had stated this in the Methods section - We ignore the helices that have none or a single charged residue. We also had a cutoff on the length of the AH as 10 - i.e. we are looking for reasonably long AHs - we had not mentioned this constraint. We have modified the Methods section to reflect this. An AH having just two similarly charged residues in a reasonably long AH (and none other) is relatively significant. For example, one charged residue in VP24 (D124) makes an electrostatic contact with human karyopherin, while the other one E113 makes a contact to Arg140 in another helix (α6) in VP24 2.The third helices described in detail are from Vps35 and the authors identify several helices with carry charges, but no clear targets are discussed.We have stated that ‘we have not been able to identify a critical role for this helix in the protein from current literature’, which does not preclude the importance of these helices. This, in fact, highlights the ability of our method to extract helices that might be of significance, yet not probed sufficiently as targets. At the same time, it is also equally possible that this helix is not functionally significant.Page 6: The authors make a connection between the number of acidic residues in ahelix from Ebola Vps35 compared to Marburg Vps35 and the frequency of outbreaks, which is a complete over interpretation of their data.We agree with this criticism, and have made the corrections.In summary the manuscript describes an interesting approach to identify or validate potential drug targets.We appreciate the positive and encouraging note on our efforts to use computational methods to identify critical regions of interaction in the Ebola proteins, which could be easily extended to other organisms as well.However, the authors need to be more cautious in interpreting their results. Without any experimental validation their approach to link helical properties to protein interaction propensities is extremely weak.We hope that we have addressed your concerns by the changes that we have made. We also expect future results to corroborate some of our predictions, and will make the updates on the f1000 site (which their format allows us to). We sincerely hope that the manuscript will be found suitable in the modified form for publication.Thanking you,Sincerely,Sandeep Chakraborty (Corresponding author) References 1. Chakraborty S: DOCLASP - Docking ligands to target proteins using spatial and electrostatic congruence extracted from a known holoenzyme and applying simple geometrical transformations [v1; ref status: awaiting peer review, http://f1000r.es/48g]. F1000Research. 2014; 3 (262). Publisher Full Text \| Reference Source 2. Chakraborty S: Correlating the ability of VP24 protein from Ebola and Marburg viruses to bind human karyopherin to their immune suppression mechanism and pathogenicity using computational methods [v1; ref status: awaiting peer review, http://f1000r.es/4o3]. F1000Research. 2014; 3 (265). Publisher Full Text \| Reference Source 3. Lee JE, Fusco ML, Hessell AJ, Oswald EB, et al.: Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor. Nature. 2008; 454 (7201): 177-182 PubMed Abstract \| Free Full Text \| Publisher Full Text \| Reference Source" } ] } ]	1	https://f1000research.com/articles/3-251
https://f1000research.com/articles/4-23/v1	23 Jan 15	{ "type": "Research Article", "title": "Levodopa effects on [11C]raclopride binding in the resting human brain", "authors": [ "Kevin J. Black", "Marilyn L. Piccirillo", "Jonathan M. Koller", "Tiffany Hseih", "Lei Wang", "Mark A. Mintun", "Marilyn L. Piccirillo", "Jonathan M. Koller", "Tiffany Hseih", "Lei Wang", "Mark A. Mintun" ], "abstract": "Rationale: Synaptic dopamine (DA) release induced by amphetamine or other experimental manipulations can displace [11C]raclopride (RAC) from dopamine D2-like receptors. We hypothesized that exogenous levodopa might increase dopamine release at striatal synapses under some conditions but not others, allowing a more naturalistic assessment of presynaptic dopaminergic function. Presynaptic dopaminergic abnormalities have been reported in Tourette syndrome (TS).Objective: Test whether levodopa induces measurable synaptic DA release in healthy people at rest, and gather pilot data in TS.Methods: This double-blind crossover study used RAC and positron emission tomography (PET) to measure synaptic dopamine release 4 times in each of 10 carbidopa-pretreated, neuroleptic-naïve adults: before and during an infusion of levodopa on one day and placebo on another (in random order). Five subjects had TS and 5 were matched controls. RAC* binding potential (BPND) was quantified in predefined anatomical volumes of interest (VOIs). A separate analysis compared BPND voxel by voxel over the entire brain.Results: DA release declined between the first and second scan of each day (p=0.012), including on the placebo day. Levodopa did not significantly reduce striatal RAC* binding and striatal binding did not differ significantly between TS and control groups. However, levodopa’s effect on DA release differed significantly in a right midbrain region (p=0.002, corrected), where levodopa displaced RAC* by 59% in control subjects but increased BPND by 74% in TS subjects.Discussion: Decreased DA release on the second scan of the day is consistent with the few previous studies with a similar design, and may indicate habituation to study procedures. We hypothesize that mesostriatal DA neurons fire relatively little while subjects rest, possibly explaining the non-significant effect of levodopa on striatal RAC* binding. The modest sample size argues for caution in interpreting the group difference in midbrain DA release with levodopa.", "keywords": [ "dopamine", "D2", "receptor", "raclopride", "positron", "emission", "tomography", "PET", "levodopa", "dopamine", "Tourette", "syndrome", "nucleus", "accumbens", "substantia", "nigra", "midbrain" ], "content": "Introduction\n\nDopamine (DA) release from neurons has often been conceptualized as occurring via two separable mechanisms: tonic, referring to low levels of DA in extrasynaptic spaces that may be more accessible to microdialysis, and phasic, referring to synaptic DA release at synapses following presynaptic action potentials1. Phasic dopamine release is crucial to dopamine’s role in changing behavior2, including in learning sequences of movements3. Normal tonic dopamine release but abnormal phasic dopamine release has been postulated to occur in several disease states, including drug abuse4 and Tourette syndrome (TS)5–8.\n\nThe radioligand [11C]raclopride (hereinafter RAC) binds to dopamine D2-like (D2, D3 and D4) receptors loosely enough to be displaced by physiological increases of dopamine at the synapse. This property has been exploited to detect changes in synaptic DA release induced by experimental manipulations including the administration of amphetamine9. However, amphetamine also has some disadvantages in this context—primarily, that it does not really produce phasic dopamine release in the usual, temporal, sense of the word. Rather, it causes prolonged, substantial dopamine release regardless of environmental demands. Scientific questions about DA release in the absence of amphetamine might be better tested with a pharmacological stimulus that could potentially increase the magnitude of DA release, but under tighter endogenous control. Additionally, amphetamine can induce symptomatic effects including euphoria10 and transiently increased tic severity11; these effects can themselves alter brain activity, complicating interpretation of the results. Ideally, a pharmacological challenge drug to test phasic dopamine release would not produce effects noticed by the subject.\n\nThe present study provides preliminary data for a novel approach to testing presynaptic dopamine release using levodopa, the body’s natural synthetic precursor to dopamine. Exogenous levodopa boosts dopamine synthesis almost immediately in both parkinsonian and healthy brains [reviewed in 12]. The extra dopamine is rapidly released at the synapse in people with DA deficiency13, and there is evidence that this happens also in the non-parkinsonian brain. In people, including in people with tics, levodopa produces dose-dependent yawning, mild sleepiness, and effects on working memory—i.e., CNS-mediated effects14–16. Additional evidence for levodopa-induced synaptic DA release in the non-parkinsonian brain is reviewed in 12. When given after an adequate dose of carbidopa, which prevents conversion to dopamine but does not cross the blood-brain barrier, systemic levodopa administration essentially delivers dopamine selectively to the brain, as confirmed by the fact that it does not alter quantitative whole-brain blood flow17–19, as dopamine would if it were being delivered systemically or produced outside the brain. In fact, with adequate carbidopa pretreatment, volunteers usually cannot tell whether they are receiving levodopa or a placebo12,16.\n\nWe used PET and RAC to measure synaptic dopamine release in response to a standardized levodopa infusion (after carbidopa) in 10 subjects. Since no previous data were available on levodopa effects on RAC* PET, we included before- and during-levodopa RAC* PET scans as well as before- and during-placebo scans. Half of the subjects had a chronic tic disorder and the other half were matched control subjects without tics, to generate preliminary data in each population. The original hypotheses were that levodopa would stimulate striatal dopamine production in the controls, but may affect people with TS differently.\n\n\nMethods\n\nThis study was approved by the Human Studies Committee of Washington University School of Medicine (IRB, protocol # 03-0347, the WUSM Radioactive Drug Research Committee (protocol # 497F), and the U.S. Food and Drug Administration (Investigator IND #69,745 for i.v. levodopa). All subjects provided written confirmation of informed consent before study participation.\n\nDiagnostic assessment included psychiatric and neurological examination by a movement-disorders-trained neuropsychiatrist (KJB) and a validated semistandardized psychiatric diagnostic interview [SCID-IV; 20]. Tic subjects met DSM-IV-TR criteria for Tourette’s Disorder. Control subjects with no history of tics were matched one-to-one for age, sex and handedness (with one ambidextrous TS subject matched to a right-handed control). Exclusion criteria included any lifetime neurological or Axis I psychiatric disorder (except that TS, ADHD and OCD were allowed in tic subjects, and migraine and specific phobia were allowed in either group), current serious general medical illness, medication history of dopamine antagonists or other drugs likely to affect the dopaminergic system, current use of any neuroactive medication, lactation, possibility of pregnancy, or contraindication to levodopa or MRI.\n\nClinical features were characterized by the Diagnostic Confidence Index (0=no features of TS; 100=all enumerated features of classic TS; scores in the original clinical validation sample ranged from 5 to 100, mean=61, S.D.=20)21; the YGTSS, an expert-rated measure of tic severity over the previous week (motor tic scale 0–25, vocal tic scale 0–25, impairment scale 0–50, higher scores indicating a higher symptom burden)22,23; the revised Tic Symptom Self-Report (TSSR) scale, a self-report scale including scores of 0–3 for each of 18 motor tics and 16 vocal tics, with 3 indicating tics were “very frequent and very forceful” over the preceding two weeks24,25; the ADHD Rating Scale, an expert-rated measure of current severity of Attention-Deficit/Hyperactivity Disorder (ADHD) based on DSM-IV criteria (range 0–54, higher scores indicating a higher symptom burden)26; and the Y BOCS, an expert-rated measure of current obsessive-compulsive disorder (OCD) severity (range 0–40, higher scores indicating a higher symptom burden)27,28.\n\nEach subject had 4 RAC* PET scans: two scans on each of two days at least a week apart (Figure 1). After oral carbidopa and the baseline PET scan, an infusion of levodopa or saline placebo was begun by vein at an individualized dose intended to produce a steady-state levodopa plasma concentration of 600ng/mL. After allowing 30 minutes to approach steady-state levodopa concentration, a second scan was done while the infusion continued. The order (levodopa on day 1 and placebo on day 2, or the reverse) was assigned randomly to each subject, and subjects and PET staff were blind to drug assignment during all scans.\n\nThe room was darkened and subjects were instructed to lie quietly in the scanner with eyes closed throughout each scan. Study staff asked subjects every 5 or 10 minutes if they were comfortable and made sure they were awake.\n\nSubjects took 200mg carbidopa by mouth at least 1 hour before levodopa infusion began. A dose of levodopa estimated to fill each subject’s volume of distribution at a target concentration of 600ng/mL was infused over 10 minutes, followed until the second PET scan of the day was completed by a maintenance infusion at a rate estimated to compensate for elimination. In prior work, these infusion rates produced a mean blood level across subjects of ~625ng/mL after 25 minutes of infusion16. On average, that concentration produces substantial motor benefit in early Parkinson disease29,30, yet this infusion method is well enough tolerated that subjects cannot reliably distinguish the levodopa and saline infusions12,16.\n\nLevodopa plasma concentration was measured by a validated method31.\n\n[11C]raclopride was prepared by O-[11C]methylation of (S)-O-desmethylraclopride HBr (ABX Advanced Biochemical Compounds, Radeberg, Germany) using a modification of previously reported procedures32,33. Carbon-11 was produced as 11CO2 using the Washington University JSW BC 16/8 cyclotron and the 14N(p,α)11C nuclear reaction. The 11CO2 was converted to 11CH3I using the microprocessor-controlled PETtrace MeI MicroLab (GE Medical Systems, Milwaukee, WI), and immediately used for [11C]methylation of (S)-O-desmethylraclopride. Product [11C]raclopride was purified via semipreparative HPLC, and reformulated in a 10% ethanol/normal saline solution. The radiochemical purity exceeded 95%, and the specific activity exceeded 500 Ci/mmol, as determined by analytical HPLC. The mass of raclopride was ≤13.9 µg per injected dose.\n\nRAC* was given i.v. over an interval of 30 seconds (median dose 14.8mCi, interquartile range 11.0–18.9mCi). PET images were acquired on a Siemens ECAT 961 camera beginning with arrival of radiotracer in the head and continuing for 60 minutes using image frames of increasing duration. An MP-RAGE sequence was used to acquire a 3-dimensional T1-weighted image of the brain with acquisition time ~400 sec and voxel dimensions 1.25×1×1mm3.\n\nThe PET images were realigned within each subject and then to the subject’s MRI using a rigid-body alignment method with low measured error, optimized for dynamic PET images34–37.\n\nNine subcortical volumes of interest (VOIs) were defined for each subject from that subject’s MRI by a high-dimensional semi-automated method of known high test-retest reliability38 (Figure 2). These VOIs corresponded to the thalamus and the left and right putamen, caudate, nucleus accumbens, and globus pallidus. An additional VOI was created from the average (weighted by region volume) of 22 FreeSurfer-labeled gray matter regions comprising frontal cortex (11 left- and 11 right-hemisphere VOIs). This large frontal VOI produced adequate counting statistics for modest noise in the time-activity curve (Figure 3). A cerebellum VOI was traced on each subject’s MR image. All VOIs were transferred to each subject’s realigned PET images using the optimized MRI-to-PET transformation matrix computed in the alignment step. The cerebellar VOI was trimmed if needed so that no voxel in the VOI corresponded to any of the inferior-most four slices in any frame of that subject’s original PET images. Thus in each subject each VOI was identical for all four PET scans.\n\nAtlas-based VOI outlines are shown on an axial section from one subject (Cd yellow, Pu light blue, Pl white, Th red; NA does not appear on this section).\n\nThe binding potential BPND39,40, an estimate of the quotient Bmax/KD, was computed as one less than the distribution volume ratio (DVR), which was derived for each of the nine subcortical VOIs and the frontal lobe VOI using the cerebellar reference region41. As we had no a priori hypothesis about laterality of results in any of the paired basal ganglia nuclei, we averaged corresponding left and right BPNDs (weighted by VOI volume) to produce for each PET scan six final BPND values, one each for frontal lobe cortex (FL), thalamus (Th), putamen (Pu), caudate (Cd), nucleus accumbens (NA), and globus pallidus (Pl).\n\nThe primary statistical analysis used a repeated-measures analysis of variance (rmANOVA) with BPND as dependent variable, diagnosis (tic or control) as a between-group variable, time (before or during the infusion) and day (placebo or levodopa) as within-subject variables, and region (the six VOI-based BPNDs) as a repeated measure. Exploratory analyses used an ANOVA for each region.\n\nFor each subject, a DVR image was computed using at each voxel in the brain the Logan graphical method with the cerebellar VOI described in the preceding section as reference region41. As a methods check, the mean across striatal VOIs of the voxelwise DVR value was essentially identical to the regional DVR computed using the standard methods described above. Analysis was limited to voxels in atlas space at which every subject contributed data from all frames of the dynamic PET acquisition.\n\nWhole-brain comparisons used voxelwise t tests corrected for multiple comparisons in SPM 8, as follows. A t test compared DVR images between the TS and the control group, and clusters of contiguous voxels with t exceeding the threshold corresponding to p<0.001 were accepted as significantly different between groups if cluster volume exceeded the threshold required to control False Discovery Rate (FDR) for the entire dataset at p<0.05.\n\nTwo comparisons were made, one based on mean baseline DVR images and the other based on levodopa effect ΔDVR images. Each subject’s two pre-infusion RAC* PET scans, one from each scan day, were averaged to create that subject’s mean baseline DVR image. The difference of the during-levodopa DVR image and the during-placebo DVR image in a subject was used to create that subject’s levodopa effect ΔDVR image.\n\n\nResults\n\nSubject characteristics and adequacy of matching are reported in Table 1, and clinical characteristics of the TS group are reported in Table 2.\n\nAbbreviations: OCD=Obsessive-compulsive disorder, ADHD=Attention Deficit Hyperactivity Disorder.\n\nThe Y BOCS was completed for only 1 tic subject; the score was 9 on day 1 and 14 on day 2\n\nAbbreviations: DCI=Tourette Syndrome Diagnostic Confidence Index, YGTSS=Yale Global Tic Severity Scale, Y-BOCS=Yale-Brown Obsessive Compulsive Scale, ADHD=Attention Deficit Hyperactivity Disorder, TSSR=Tic Symptom Self Report.\n\nLevodopa plasma concentrations were ~800–1000ng/ml before the RAC* scan and ~500–700ng/ml after the RAC* scan, and did not differ significantly between groups (Table 3).\n\nThe a priori VOIs showed higher and more reliable binding in striatum and pallidum, as expected. Nevertheless, the thalamus, GP and frontal cortex VOIs also produced good counting statistics (Figure 3). For every one of the VOIs the baseline BPND estimates were positive in all 120 scans, and were very similar between the two scan days (Table 4, Figure 4).\n\nDecay-corrected time-activity curves are shown for the right putamen (filled circles), the frontal lobe VOI (+’s), and the cerebellar reference region (empty circles) from one subject’s pre-levodopa PET scan.\n\nBPNDs from the first scan of each day are plotted for all 10 subjects, with the BPND from the pre-placebo scan on the horizontal axis and from the pre-levodopa scan on the vertical axis. For the paired VOIs the mean of the left and right BPND is used. The diagonal line is the line of identity. The inset shows an enlarged view of the data from the frontal lobe and thalamus VOIs.\n\nAbbreviations: FL, frontal lobes; Th, thalamus; Pl, pallidum; NA, nucleus accumbens; Cd, caudate; Pu, putamen.\n\nThis study includes a before- and after-infusion scan on each of two days. On one day the infusion contains levodopa, and on the other day it is a saline placebo. Thus each subject has three non-levodopa scans (the first scan of each day plus the scan during the placebo infusion). As expected, BPND was quite reproducible in the two pre-levodopa scans (correlated at r = 0.99 across VOI and subject).\n\nTo our surprise, BPND increased between the 1st and 2nd scan of the day (main effect of time, F=10.605, df=1,8, p=0.012), and this change did not differ significantly between the levodopa and placebo days (time × day interaction, F=0.014, df=5,4, p=0.909). In other words, the two scans on the placebo day were not identical. Mean BPND was 2.7% to 24.0% higher during the placebo infusion, indicating decreased dopamine release compared to earlier on the same day. The change from the first to the second scan of each day was significant in most individual region analyses: main effect of time, thalamus p=0.002, frontal lobe p=0.032, caudate p=0.039, pallidum p=0.048, and nucleus accumbens p=0.052 (multivariate time × region interaction F=4.173, df=5,4, p=0.096). Figure 5 shows the BPND for each VOI from both scans on the placebo day only.\n\nFor each of the a priori VOIs, mean BPND across all 10 subjects is shown before and during the infusion on the placebo day only. Error bars show SD. Numeric labels are p values for the main effect of time in the individual region ANOVAs (putamen p=.115).\n\nSince the pre- and on-placebo scans differed, the appropriate comparison for the on-levodopa RAC* scan is the on-placebo scan. Therefore we assessed the effect of levodopa by comparing the BPND in the on-LD and on-placebo scans. In the VOI analysis, there was no significant effect of LD (day × time interaction, F=0.014, df=1,8, p=0.909).\n\nTS vs control at baseline. For the ANCOVA across all regions, RAC* binding did not differ significantly between tic and control subjects (main effect of diagnosis, F=0.744, df=1,8, p=0.413; tic vs control). Nevertheless, baseline RAC* binding was numerically higher in TS by 13–17% in the three striatal VOIs and by 5–7% in the frontal lobe and thalamus VOIs. The whole-brain analysis identified no significant regional differences in baseline RAC* binding between TS and control subjects.\n\nTS vs control: time effect (change from first to second scan). There was a trend for the change in BPND during the infusion to be smaller in tic subjects (time × diagnosis interaction F=4.211, df=1,8, p=0.074). Each of the three striatal regions showed a similar effect when analyzed individually (0.05 < p < 0.10). Figure 6 shows the VOI BPND values before and during the placebo infusion, by diagnosis.\n\nMean BPNDs from the a priori VOIs, before and during the infusion on the placebo day only. Error bars show SD. The p values shown are for the time × diagnosis interaction in the individual region ANOVAs.\n\nTS vs control: effect of levodopa on RAC* binding. In the a priori VOIs, the effect of LD did not differ overall in tic subjects (day × time × diagnosis interaction, F=1.308, df=1,8, p=0.286), and the 4-way interaction (day × time × diagnosis × region) was not significant (F=1.577, df=5,4, p=0.340). Although not statistically significant, pallidal and thalamic BPND tended to decrease in control subjects but increase in the tic subjects (Figure 7).\n\nMean BPND for the a priori VOIs is shown during the levodopa and placebo infusions; the error bar indicates SD. The day × time × diagnosis interaction and the day × time × diagnosis × region interaction were not significant. The daggers indicate a trend in the thalamic and pallidal VOIs for BPND to decrease with levodopa in the control group but increase with levodopa in the tic group (regional ANOVA, day × time × diagnosis interaction, pallidum p=0.050, thalamus p=0.098).\n\nThe whole-brain analysis identified a similar but statistically significant effect in two clusters, where RAC* binding decreased with levodopa in controls, consistent with increased dopamine release during the levodopa infusion, but RAC* binding increased in the TS group. The first cluster included 38 voxels in midbrain (1.0 ml, FDR corrected p=0.002), with a peak t value of 9.0 (8 df) at atlas coordinate (1.5, −21, −15) and extending laterally in approximately the right substantia nigra/ventral tegmental area (Figure 8a). A second significant cluster of 19 voxels (0.5 ml, corrected p=0.023) occurred in parahippocampal gyrus, with peak t=7.92 at (22.5, −39, −6) (Figure 8b). The mean change in BPND with levodopa in these regions is shown in Figure 8c. In both these clusters, the BPND on placebo was positive in all subjects (p < 0.001, binomial distribution), consistent with nontrivial RAC* binding. The highest t value in the whole-brain comparison, 11.62, occurred in Brodmann’s area 13, but the cluster volume was only 0.1 ml, not significant by FDR correction (Figure 8d).\n\nDifferences in the RAC* binding response to levodopa between TS and control subjects, thresholded at uncorrected p = 0.001, in color, laid over the MRI template image in grayscale. a, b: Significant clusters, with blue lines crossing at the peak t value in midbrain (a, three views) and in parahippocampal gyrus (b). A third statistically significant cluster was centered at the posterior edge of the occipital lobe, but both the location and the observation that in this cluster the BPND on placebo was negative in half the subjects suggests that this cluster likely does not reflect specific binding. c: Levodopa-induced change in BPND, TS vs. control, in the clusters shown in A and B. R., Right; PHG, parahippocampal gyrus. Asterisks indicate that mean BPND differs significantly from zero. d: The blue lines cross at the voxel with the highest t value in the whole-brain SPM analysis of levodopa effect ΔDVR images (t=11.62, 8 df).\n\n\nDiscussion\n\nBPND increased from before to during the placebo infusion in the striatum, thalamus and frontal lobe VOIs, especially in control subjects (Figure 5, Figure 6). Surprisingly little information describes within-day stability of RAC* binding, though several studies compare binding across time intervals of days to months42–45. Mawlawi et al.46 scanned 10 subjects twice each on the same day using a bolus-plus-constant-infusion method, and found no significant mean change from the first to the second scan. However, Alakurtti and colleagues47 found that mean BPND increased from the first to the second scan of the day in striatal and thalamic regions, with the change (about +5%) reaching statistical significance in medial and lateral thalamus.\n\nThe observation in the present study that BPND increased from the first to second scan of the day is consistent with this background, and is relevant to RAC* challenge PET studies in general, because essentially all such studies use a before- vs. after-intervention design. Slifstein et al. [48, p. 357] argue that the existence of placebo-induced DA responses make the before-after model more appropriate for amphetamine challenge studies. However, our results and those of Alakurtti et al.47 suggest that BPND increases from the first to the second scan even without active intervention. This does not invalidate the results of most before-after RAC* studies, since amphetamine challenge decreases striatal RAC* BPND by a large fraction, and to a lesser extent so do many cognitive and behavioral interventions in such studies, including studies of the placebo effect. However, the present results suggest that before-after RAC* studies may be less sensitive to manipulations that would decrease dopamine release.\n\nPossible pathophysiological interpretation. The increase in BPND during the placebo infusion is most likely associated with passage of time rather than a placebo effect per se, especially as placebo administration is more likely to increase dopamine release48–50. The presumed decrease in dopamine release during the placebo infusion could indicate that control subjects accommodate to the scanner environment over the course of the study day.\n\nLevodopa effect on RAC* binding in striatum. Striatal RAC* binding was not substantially changed by levodopa. Initially this result came as a surprise to the authors, because levodopa was given expressly with the expectation that it would increase synaptic dopamine levels. Briefly, support for this expectation includes the following. First, in Parkinson disease there is overwhelming evidence both by clinical observations and by RAC* PET imaging that exogenous levodopa substantially increases striatal dopamine release51–53. But there is also evidence in subjects without dopamine deficiency: intravenous levodopa is rapidly taken up from the bloodstream into the brain and converted into dopamine, and several studies show that it then boosts synaptic dopamine release [reviewed in 12]. For instance, exogenous levodopa produces clear sedative and cognitive effects in healthy people54–56. Thus the authors originally expected that exogenous levodopa would decrease striatal RAC* binding.\n\nHowever, further reflection and reading have motivated a different view whereby the results support the original goal of choosing a pharmacological challenge agent that would stimulate phasic dopamine release, but under endogenous control. Recall that the concern with stimulants as challenge agents was that they cause a substantial release of dopamine at the striatal synapse regardless of current environmental demands; this approach may produce a ceiling effect for dopamine release that does not reflect typical endogenous control. A sensible hypothesis to explain the results of the present study would be that a research subject lying awake in a quiet, darkened room without specific cognitive demands has no need for substantial phasic release of dopamine, and thus even if exogenous levodopa has added dopamine to presynaptic vesicles, they are not released at a substantial rate at the synapse. A levodopa-raclopride study of a motor task in healthy individuals provides direct experimental support of this hypothesis57. That study was properly designed with two sessions, placebo on one day and levodopa on another, with randomized order. Levodopa increased striatal dopamine release during performance of a motor task, but not at rest. Since in the present study all subjects were at rest during all scans, the results are consistent with those of Flöel and colleagues57.\n\nThe tic and control subgroups have only five subjects each, and differences between the tic and control groups in the a priori VOIs were not statistically significant, so there is little need to comment further on these results. Previous drafts of this report included such discussion58.\n\nThe whole-brain analysis comparing RAC* binding with levodopa vs. placebo did identify statistically significant differences (Figure 8a–c). In midbrain (approximately substantia nigra/VTA) and in parahippocampal gyrus, levodopa stimulated dopamine release in controls but reduced it in TS subjects in. A similar pattern, though not statistically significant, was observed in orbital cortex (Brodmann’s area 13), thalamus and globus pallidus (Figure 7 and Figure 8d).\n\nOne expects exogenous levodopa to increase dopamine release in the substantia nigra, as occurred in the control subjects. D2 and D3 dopamine receptors are present in the substantia nigra and their activation inhibits spike firing, dopamine synthesis and dopamine release by nigral dopaminergic cells59. We hypothesize that levodopa increased dopamine stimulation of these inhibitory D2-like receptors in control subjects, and this may have prevented levodopa from stimulating nigrostriatal dopamine release into the striatum.\n\nSubjects with TS, however, showed an increase in substantia nigra RAC* binding with levodopa, consistent with a decrease in nigral dopamine release. Nigral dopamine release has been related to reward and novelty in humans. Healthy adults with higher novelty seeking scores had lower D2-like binding ([18F]fallypride) in SN, consistent with greater dopamine release60. Functional MRI studies have also demonstrated substantia nigra signal related to stimulus novelty or to the Novelty Seeking trait61–63. Healthy adults receiving a sweet vs salty taste had BOLD activation in this region64. Despite this information, it is not clear how to relate a decrease in levodopa-stimulated dopamine release in substantia nigra to the pathophysiology of TS. Explaining the similar difference in nigral levodopa response in TS in parahippocampal gyrus and orbital cortex is no easier, though dopaminergic effects on D2-like binding in hippocampus have been documented in Parkinson disease65 and dopamine agonists evoke changes in orbital cortex activity66. The trend for a similar effect in thalamus is consistent with a [11C]FLB-457 PET study in which amphetamine provoked thalamic dopamine release in control subjects but not in TS67. Overall, these results are consistent with an abnormality of presynaptic dopaminergic pharmacology in TS, but the limitations of this comparison must be acknowledged.\n\nHigher-affinity radioligands, such as [18F]fallypride or [11C]FLB 457, have advantages for measuring cortical D2Rs, e.g. in the frontal lobe where D2Rs appear at much lower concentrations than in the striatum. There are two primary concerns with RAC* outside the striatum [reviewed thoroughly in 9]. The first concern is a reliability issue: since the concentration of D2-like receptors is low in cortex compared to striatum, the counting statistics are poor for cortical VOIs of similar volume, and this renders the computed BPNDs suspect. For instance, some regional RAC* BPNDs are negative or close enough to zero that displacement studies produce results that are hard to interpret. In the present study, FreeSurfer-defined cortical regions allowed the creation of a large, reliably defined frontal lobe VOI, in which PET time-activity curves were low in noise (Figure 3), allowing statistically reliable estimates of BPND that were uniformly positive (Table 4, Figure 4). Similarly RAC* displacement in thalamus has shown adequate counting statistics and reliability in previous studies47,68.\n\nThe second concern with RAC* in extrastriatal regions is one of validity or interpretation. RAC* binding in cortex includes some nonspecific binding33, so a fair question is to what extent specific binding in cortex represents dopamine D2-like receptors. D2 and D4 receptors are expressed in human prefrontal cortex, though at relatively low concentrations compared to striatum69. On the other hand, at least one study’s results suggest that raclopride may have superior sensitivity to fallypride for measuring dopamine release in some cortical regions70. The validity concern is less worrisome in human thalamus, which contains predominantly D3 rather than D2 receptors71, and in substantia nigra, where D2 and D3 receptors are well characterized. Other authors have interpreted substantia nigra RAC* displacement as indicating synaptic dopamine release9.\n\nFinally, comparing TS and control subgroups of only five subjects each provides insufficient power to identify some true group differences (type II error). More importantly, the small sample size lowers confidence in how representative the statistically significant differences are of the overall population of adults with TS.\n\nThese results suggest that a natural next step for research in TS is to test whether dopamine release in TS differs during a dopamine-releasing cognitive (or other) task. Levodopa may augment the task-evoked release or interact with it differently in people with versus without tics. Along these lines, a cognitive-pharmacological interaction fMRI study in TS found that LD changed the BOLD responses to a working memory task72. A newer levodopa infusion method produced roughly twice as high a levodopa plasma concentration as the infusion used in this study12, and may produce greater dopamine release.\n\n\nData availability\n\nF1000Research: Dataset 1. PET images and clinical data, 10.5256/f1000research.5672.d4217274\n\n\nConsent\n\nAll subjects provided written confirmation of informed consent before study participation.", "appendix": "Author contributions\n\n\n\nDesigned study: KJB\n\nAuthorized User, i.e. responsible for appropriate human administration of radiopharmaceuticals: MAM\n\nAnalyzed data: KJB, MLP, JMK, TH, LW, MAM\n\nContributed research tools: JMK, LW, MAM\n\nSearched and summarized relevant literature: MLP\n\nWrote the manuscript: KJB\n\nReviewed drafts and approved the final draft: KJB, MLP, JMK, TH, LW, MAM\n\n\nCompeting interests\n\n\n\nAuthor KJB received honoraria for educational presentations from a grant from the US CDC to the Tourette Syndrome Association. There are no other potential conflicts of interest.\n\n\nGrant information\n\nData collection was supported by the Tourette Syndrome Association and manuscript preparation was supported in part by NIH grants K24 MH087913 and R21 MH098670.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nThe authors gratefully acknowledge recruitment assistance from the Tourette Syndrome Association, editorial suggestions from Tamara Hershey, Ph.D., and technical assistance from Johanna M. Hartlein, R.N., M.S.N., Stephen Moerlein, Ph.D., BCNP, Susan Loftin, Kathryn I. Alpert, B.A., Meghan C. Campbell, Ph.D., Kathryn Vehe, Pharm.D., Michael P. McEvilly. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFlöel A, Garraux G, Xu B, et al.: Levodopa increases memory encoding and dopamine release in the striatum in the elderly. Neurobiol Aging. 2008; 29(2): 267–279. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlack KJ, Piccirillo ML, Koller JM, et al.: Levodopa-stimulated dopamine release in Tourette syndrome. PeerJ PrePrints. 2013; 1: e30. Publisher Full Text\n\nGrace AA: Dopamine. In; Davis KL, Charney D, Coyle JT, Nemeroff C, eds. Neuropsychopharmacology: The Fifth Generation of Progress. Philadelphia, PA: Lippincott Williams & Wilkins, 2002: 2080. Reference Source\n\nZald DH, Cowan RL, Riccardi P, et al.: Midbrain dopamine receptor availability is inversely associated with novelty-seeking traits in humans. J Neurosci. 2008; 28(53): 14372–14378. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBunzeck N, Duzel E: Absolute coding of stimulus novelty in the human substantia nigra/VTA. Neuron. 2006; 51(3): 369–379. PubMed Abstract \| Publisher Full Text\n\nKrebs RM, Schott BH, Duzel E: Personality traits are differentially associated with patterns of reward and novelty processing in the human substantia nigra/ventral tegmental area. Biol Psychiatry. 2009; 65(2): 103–110. PubMed Abstract \| Publisher Full Text\n\nKrebs RM, Heipertz D, Schuetze H, et al.: Novelty increases the mesolimbic functional connectivity of the substantia nigra/ventral tegmental area (SN/VTA) during reward anticipation: Evidence from high-resolution fMRI. Neuroimage. 2011; 58(2): 647–655. PubMed Abstract \| Publisher Full Text\n\nO’Doherty JP, Deichmann R, Critchley HD, et al.: Neural responses during anticipation of a primary taste reward. Neuron. 2002; 33(5): 815–826. PubMed Abstract \| Publisher Full Text\n\nBohnen NI, Gedela S, Herath P, et al.: Selective hyposmia in Parkinson disease: association with hippocampal dopamine activity. Neurosci Lett. 2008; 447(1): 12–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlack KJ, Hershey T, Koller JM, et al.: A possible substrate for dopamine-related changes in mood and behavior: prefrontal and limbic effects of a D3-preferring dopamine agonist. Proc Natl Acad Sci U S A. 2002; 99(26): 17113–17118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSteeves TD, Ko JH, Kideckel DM, et al.: Extrastriatal dopaminergic dysfunction in tourette syndrome. Ann Neurol. 2010; 67(2): 170–181. PubMed Abstract \| Publisher Full Text\n\nHirvonen J, Aalto S, Lumme V, et al.: Measurement of striatal and thalamic dopamine D2 receptor binding with 11C-raclopride. Nucl Med Commun. 2003; 24(12): 1207–1214. PubMed Abstract\n\nMeador-Woodruff JH, Damask SP, Wang J, et al.: Dopamine receptor mRNA expression in human striatum and neocortex. Neuropsychopharmacology. 1996; 15(1): 17–29. PubMed Abstract \| Publisher Full Text\n\nSlifstein M, Kegeles LS, Xu X, et al.: Striatal and extrastriatal dopamine release measured with PET and [(18)F] fallypride. Synapse. 2010; 64(5): 350–362. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSun J, Xu J, Cairns NJ, et al.: Dopamine D1, D2, D3 receptors, vesicular monoamine transporter type-2 (VMAT2) and dopamine transporter (DAT) densities in aged human brain. PLoS One. 2012; 7(11): e49483. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHershey T, Black KJ, Hartlein JM, et al.: Cognitive-pharmacologic functional magnetic resonance imaging in Tourette syndrome: a pilot study. Biol Psychiatry. 2004; 55(9): 916–925. PubMed Abstract \| Publisher Full Text\n\nBlack KJ, Koller JM, Campbell MC, et al.: Levodopa-stimulated dopamine release in Tourette syndrome. Movement Disorders. 2010; 25: S373. Reference Source\n\nBlack KJ, Piccirillo ML, Koller JM, et al.: PET images and clinical data. F1000Research. 2015. Data Source" }	[ { "id": "7601", "date": "17 Mar 2015", "name": "Marie Vidailhet", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors studies the raclopride binding (displacement) in groups of subjects (normal and Tourette syndrome). They studied the effect of levodopa infusions and of a placebo. The subjects were studied at rest.Basically, they found that in Tourette syndrome, dopamine release was smaller (reduced) than in controls, in midbrain (approximately substantia nigra/VTA) and in parahippocampal gyrus. This is an interesting paper and the methodology is adequate. The subjects are studied at rest, this may underestimate the dynamic of dopamine release and it would have be more interesting to study this phenomenon during a task. The groups are very small, and the effect in Tourette syndrome may also be different according to the characteristics of the patient (with or without additional behavioral disorders).Nevetherless, the study is consistent with the presence of abnormality of presynaptic dopaminergic pharmacology in Tourette syndrome.", "responses": [] }, { "id": "7821", "date": "23 Mar 2015", "name": "W.R. Wayne Martin", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a carefully performed study that presents a novel approach to measure presynaptic dopamine release using the administration of exogenous levodopa, coupled with raclopride PET scanning. Preliminary data are provided using this method in a small group of controls and subjects with Tourette syndrome. The authors describe a decline in dopamine release in striatum, thalamus and frontal lobe between the first and second scan of each day in response to placebo administration in normal subjects, possibly due to habituation to study procedures. Levodopa administration did not alter striatal dopamine release differently in Tourette syndrome vs. controls. However, dopamine release differed significantly in the midbrain and parahippocampal gyrus in the two conditions. Levodopa stimulated dopamine release in controls but reduced it in Tourette subjects.Although these are important observations, the number of subjects studied was small. Hence, these must be considered pilot data although they are consistent with a rather complex dopaminergic role in Tourette syndrome. Of interest for future studies would be the evaluation of task-evoked dopamine release in response to cognitive tasks. Lastly, the observation that habituation occurs in response to placebo infusions has important implications to the interpretation of placebo-controlled studies of dopamine release.", "responses": [] }, { "id": "8060", "date": "23 Mar 2015", "name": "Lars Nyberg", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe most novel aspect of the study was the investigation of levodopa. This is very interesting. No significant levodopa effects were, however, observed. The authors offer some interesting thoughts on the reason for this null effect, most critically pointing to a need to have an active task PET design. This is a plausible argument that may stimulate further research on this topic.A potentially interesting methodological contribution is the observation of a difference between the 1st and 2nd scan on each day of scanning.The study is likely underpowered, in particular for any group comparison (5 TS, 5 ctrls), so the repeated-measures analysis was most likely not very sensitive. I would treat the observed differences between TS and control groups from the whole-brain analysis as preliminary.I may have missed it, but I could not find information about how the PET scans were reconstructed.", "responses": [ { "c_id": "1280", "date": "24 Mar 2015", "name": "Kevin J Black", "role": "Author Response F1000Research Advisory Board Member", "response": "The authors thank Prof. Nyberg for the thoughtful review. The [11C]raclopride PET data were collected in 3D mode and reconstructed on the Siemens ECAT 961 scanner console using filtered backprojection (ramp filter), with attenuation measured before each emission scan using an external 68Ge/68Ga source.We agree with all the reviewers that the between-group comparison is useful primarily as pilot data because of the small group sizes." } ] } ]	1	https://f1000research.com/articles/4-23
https://f1000research.com/articles/3-228/v1	29 Sep 14	{ "type": "Research Article", "title": "A novel mouse model of creatine transporter deficiency", "authors": [ "Laura Baroncelli", "Maria Grazia Alessandrì", "Jonida Tola", "Elena Putignano", "Martina Migliore", "Elena Amendola", "Cornelius Gross", "Vincenzo Leuzzi", "Giovanni Cioni", "Tommaso Pizzorusso", "Maria Grazia Alessandrì", "Jonida Tola", "Elena Putignano", "Martina Migliore", "Elena Amendola", "Cornelius Gross", "Vincenzo Leuzzi", "Giovanni Cioni", "Tommaso Pizzorusso" ], "abstract": "Mutations in the creatine (Cr) transporter (CrT) gene lead to cerebral creatine deficiency syndrome-1 (CCDS1), an X-linked metabolic disorder characterized by cerebral Cr deficiency causing intellectual disability, seizures, movement and behavioral disturbances, language and speech impairment ( OMIM #300352).CCDS1 is still an untreatable pathology that can be very invalidating for patients and caregivers. Only two murine models of CCDS1, one of which is an ubiquitous knockout mouse, are currently available to study the possible mechanisms underlying the pathologic phenotype of CCDS1 and to develop therapeutic strategies. Given the importance of validating phenotypes and efficacy of promising treatments in more than one mouse model we have generated a new murine model of CCDS1 obtained by ubiquitous deletion of 5-7 exons in the Slc6a8 gene. We showed a remarkable Cr depletion in the murine brain tissues and cognitive defects, thus resembling the key features of human CCDS1. These results confirm that CCDS1 can be well modeled in mice. This CrT−/y murine model will provide a new tool for increasing the relevance of preclinical studies to the human disease.", "keywords": [ "creatine transporter", "mouse model", "creatine deficiency syndrome 1" ], "content": "Introduction\n\nThe creatine (Cr) transporter (CrT, alias CRTR, MGC87396, CT1, SLC6A8, OMIM 300036) deficiency (CCDS1, OMIM #300352) is an X-linked inherited metabolic disorder characterized by cerebral Cr deficiency which results in intellectual disability, language and speech impairment, seizures and movement and behavioral disturbances, and affects about 1% of males with non- syndromic mental disability (van de Kamp et al., 2014). CrT loss of function is mostly caused by missense mutations and small deletions which are concentrated in the transmembrane domains 7 and 8 of the protein (van de Kamp et al., 2014). In physiological conditions, about half of our normal Cr requirement is satisfied by the diet. De novo endogenous synthesis of Cr takes place mainly in the kidney, liver and pancreas and involves the enzymes l-arginine: glycineamidinotransferase (AGAT) and S-adenosyl-l-methionine:N-guanidinoacetatemethyltransferase (GAMT) (Wyss & Kaddurah-Daouk, 2000). Cr is a polar hydrophilic molecule unable to cross the lipidic membranes, which uses a Na+- and Cl−- dependent plasma membrane CrT to enter the cells (Nash et al., 1994). CrT is widely expressed in the brain tissue with a prominent presence in the cortical and subcortical regions involved in motor and sensory processing, learning and memory, and regulation of emotion-related behavior (Lowe et al., 2014; Mak et al., 2009).\n\nPatients affected by cerebral creatine deficiency syndrome-1 (CCDS1) share depletion of brain Cr and the clinical phenotype with patients carrying the other two defects of Cr metabolism which involve mutations of genes encoding the biosynthesizing enzymes AGAT and GAMT (Item et al., 2001; Stockler et al., 1994). Replenishment of the brain Cr pool is the only effective therapy for Cr deficiency diseases (Battini et al., 2002; Schulze et al., 2001; Stockler et al., 1996). Unfortunately, in CCDS1 patients even very high doses of Cr, alone or combined with the Cr precursors arginine and glycine to stimulate endogenous Cr synthesis, fail to restore the Cr content in brain (Chilosi et al., 2008; Valayannopoulos et al., 2012). There have been attempts to normalize the levels of Cr in the brain with Cr-lipophilic analogs, but these compounds have proven ineffective when administered to patients (Fons et al., 2010). Thus, CCDS1 is still missing an effective treatment.\n\nPreclinical animal models are crucial tools to dissect disease pathogenic mechanisms and develop new therapeutic strategies. Only two murine models of CCDS1 are available so far, and they have only been analyzed at the behavioral and neurochemical level. An ubiquitous CrT knockout mouse model has been generated by deletion of 2–4 exons in the Slc6a8 gene. Learning and memory deficits, impaired motor activity and Cr depletion in brain and muscles have been reported in animals at three-four months of age (Skelton et al., 2011). Another murine model is based on the use of the CaMKII promoter to drive Cre-recombinase expression, achieving a CrT deletion only in postnatal forebrain excitatory neurons. This strategy was successful in avoiding the peripheral Cr depletion and the motor deficits shown by germline CrT knockout mouse. Behavioral analysis in mice at 12 months of age revealed learning and memory impairments that could be ameliorated by supplementation of cyclocreatine, a Cr analog (Kurosawa et al., 2012).\n\nFor translational studies, the phenotype variations observed in different mouse models, carrying similar mutations and the effects of genetic backgrounds highlight the importance of validating phenotypes and therapeutic efficacy in multiple models and in different laboratories (Katz et al., 2012). Such validation will hopefully increase the relevance of preclinical studies to the human disease. To increase the number of CCDS1 models, we generated a novel murine model of CCDS1 obtained by ubiquitous deletion of 5–7 exons in the Slc6a8 gene. These mice presented a remarkable Cr depletion in the brain tissue and displayed cognitive defects resembling the key features of human CCDS1, and providing a new promising CCDS1 animal model.\n\n\nMaterials and methods\n\nA Cre-conditional allele of Slc6a8 has been produced by introducing the loxP sites flanking exon 5–7 of the gene in embryonic stem (ES) cells via homologous recombination (vector PRPGS00081_A_A09 obtained from the NIH Knock-out Mouse Program, KOMP). The presence of lox sites has been checked by sequencing (sequencing service by MWG, Germany). The plasmid was linearized with NruI before electroporation into ES cells (129/Sv x C57BL/6N, clone A8, gift of A. Wutz, Wellcome Trust Centre for Stem Cell Research, Stem Cell Institute, University of Cambridge). G418-resistant clones were identified and screened by long-range PCR (Applied Biosystems Gene AMP PCR system 2700). Hybridization with a specific probe for the 5′ and 3′ arms was used to confirm the PCR results. Two independent positive ES cell clones were injected into C57BL/6N host embryos using a piezo-drill assisted 8-cell stage injection procedure developed at EMBL, Monterotondo Italy. Four out of five offspring (all >95% ES cell derived) provided germline transmission. Germline transmission of the allele was confirmed by long-range PCR and the neomycin selection cassette was removed by crossing with FLP recombinase expressing mice (Farley et al., 2000). Germline knockout mice were produced by crossing the constitutive allele to the HPLRT::Cre recombinase deleter mouse (Tang et al., 2002; Figure 1).\n\nA targeting vector was obtained from KOMP to generate mice carrying a floxed allele. Crossing these mice with a Flp deleter mouse line produced a conditional KO mouse line (cKO allele). Crossing this line with a line expressing Cre-recombinase in the germline produced the Slc6a8 null mouse used in this study (KO allele). 1F, 1R, 2F, 2R, 3F, 3F’, 3R, 4F, 4R report the sites targeted by the PCR primers to assess allele presence.\n\nAnimals were maintained at 22°C under a 12-h light–dark cycle. Food and water were available ad libitum. All experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and were approved by the Italian Ministry of Health (authorization number 147/2014-B). All necessary efforts were made to minimize both stress and the number of animals used. As CrT deficiency is an X-linked pathology and only males are consistently affected, we focused our study on male animals. Young adult males (postnatal day P40 at the beginning of testing) of each genotype (CrT–/y mutants and CrT+/y wild-type littermates) were used in behavioral experiments, while a separate group of animals (P30) was assigned to Cr level assay.\n\nGenomic DNA was isolated from mouse tail using a kit, and the protocol suggested by the manufacturer (DNeasy Blood & Tissue Kit, Qiagen, USA). DNA was amplified for mutant and wild-type (WT) allele using a standard PCR protocol with the following primers: F:AGGTTTCCTCAGGTTATAGAGA; R:CCCTAGGTGTATCTAACATCT; R1: TCGTGGTATCGTTATGCGCC. For PCR amplification we used 300 ng of DNA in a 25 μL reaction volume containing 0.2 mM of each dNTP, 2 μM of F primer, 1 μM of R, 1 μM of R1 primer and 0.5 U/μL Red Taq DNA polymerase (Sigma-Aldrich, Italy). The PCR conditions were as follows: 94°C for 4 min followed by 37 cycles at 94°C for 30 s, 58°C for 30 s, 72°C for 40 s and a final extension at 72°C for 7 min. Amplicons were separated using 2% agarose gel and visualized under UV light after staining with Green Gel Plus (Fisher Molecular Biology, Rome, Italy). Amplicon sizes were: WT allele = 462 bp; mutant allele = 371 bp.\n\nMouse tissues, immediately frozen on dry ice and stored at -80°C until the analysis, were homogenized in 0.7 ml PBS buffer (Sigma-Aldrich, Italy) at 4°C using a ultrasonic disruptor (Microson Heat System, NY, USA) for brain or a glass manual homogenizer (VWR, Italy) for kidney, heart and muscle. After centrifugation (600 × g for 10 min at 4°C) an aliquot of the homogenate (50 µl) was assayed for protein content (Lowry et al., 1951), and the supernatant used for Cr assay as previously described (Alessandrì et al., 2005). Briefly, 50 µl of saturated sodium hydrogen carbonate and 50 µl of a mixture containing 2- phenylbutyric acid (I.S.) in toluene (6.09 mmol/l; Sigma-Aldrich, Italy) were added to 200 µl of homogenate. After adding 1 ml of toluene and 50 µl of hexafluoro-2,4-pentanedione (Sigma-Aldrich, Italy) to form bis-trifluoromethyl- pyrimidine derivatives, the mixture was stirred overnight at 80°C. The organic layer was centrifuged, dried under nitrogen and 2 µl of the residue derivatized at room temperature with 100 µl of BSTFA+TMCS (Sigma-Aldrich, Italy) injected into the GC/MS. GC analyses were performed using an Agilent 6890N GC equipped with an HP5MS capillary column (0.25 mm × 30 m, film thickness 0.25 ìm) and an Agilent mass spectrometer 5973N (Agilent Technologies, Italy). The mass spectrometer was set in EI- single ion monitoring mode (SIM). The ions with m/z of 192 for I.S., 258 for Cr and 225 for guanidinoacetic acid (GAA) were used for calculation of the metabolites, using standard curves ranging 5–90 µmol/L and 0.30–6 µmol/L for Cr and GAA, respectively. Data were processed by the G1701DA MSD ChemStation software. All the aqueous solutions were prepared using ultrapure water produced by a Millipore system.\n\nThe testing order consisted of: open field (1 day duration), object recognition test (ORT) at 24h (3 days), Y maze (1 day), Morris water maze (MWM) with hidden platform (7 days), and locomotor activity (1 day). The mice were tested on one task at a time with the next behavioral test starting at least 2 days after the completion of the previous one. In order to reduce the circadian effects, all behavioral tests were performed during the same time interval each day (1400–1800h; light phase). All behavioral tests were conducted in blind with respect to the genotype of animals. Mice were weighed at the end of experiments (P60).\n\nWe followed the protocol reported in Lonetti et al., 2010. Briefly, the apparatus consisted of a square arena (60 × 60 × 30 cm) constructed in poly(vinyl chloride) with black walls and a white floor. The mice received two sessions of 10-min duration in the empty arena on two consecutive days to habituate them to the apparatus and test room. Animal position was continuously recorded by a video tracking system (Noldus Ethovision XT). In the recording software an area corresponding to the center of the arena (a central square 30 × 30 cm), and a peripheral region (corresponding to the remaining portion of the arena) were defined. The total movement of the animal and the time spent in the center or in the periphery area were automatically computed. The mice activity during the first day of habituation was analyzed for evaluating the behavior in the open field arena. The ORT consisted of two phases: sample and testing phase. During the sample phase, two identical objects were placed in diagonally opposite corners of the arena, approximately 6 cm from the walls, and mice were allowed 10 min to explore the objects, then they were returned to their cage. The objects to be discriminated were made of plastic, metal, or glass material and were too heavy to be displaced by the mice. Arena and objects were cleaned with 10% ethanol between trials to stop the build-up of olfactory cues. The testing phase was performed 24h after the sample phase. One of the two familiar objects was replaced with a new one, while the other object was replaced by an identical copy. The objects were placed in the same locations as the previous ones. The mice were allowed to explore objects for 5 min. To avoid possible preferences for one of two objects, the choice of the new and old object and the position of the new one were randomized among animals. The amount of time spent exploring each object (nose sniffing and head orientation within <1.0 cm) was recorded and evaluated by the experimenter blind to the mouse genotype. Mice exploring the two objects for less than 10 s during the sample phase were excluded from testing. A discrimination index was computed as DI = (Tnew - Told)/(Tnew + Told), where Tnew is the time spent exploring the new object, and Told is the time spent exploring the old one.\n\nSpontaneous alternation was measured using the Y-maze, as described in Begenisic et al., 2014. We used a Y-shaped maze with three symmetrical grey solid plastic arms at a 120-degree angle (26 cm length, 10 cm width, and 15 cm height). Mice were placed in the center of the maze and allowed to freely explore the maze for 8 minutes. The apparatus was cleaned with 10% ethanol between trials to avoid the build-up of odor traces. All sessions were video-recorded for offline blind analysis. The arm entry was defined as all four limbs within the arm. A triad was defined as a set of three arm entries, when each entry was to a different arm of the maze. The number of arm entries and the number of triads were recorded in order to calculate the alternation percentage (generated by dividing the number of triads by the number of possible alternations and then multiplying by 100).\n\nMice were trained for four trials per day and for a total of 7 days in a circular water tank, made from grey polypropylene (diameter, 120 cm; height, 40 cm), filled to a depth of 25 cm with water (23°C) rendered opaque by the addition of a small amount of a non-toxic white paint. Four positions around the edge of the tank were arbitrarily designated North (N), South (S), East (E), and West (W), which provided four alternative start positions and also defined the division of the tank into four quadrants, i.e., NE, SE, SW, and NW. A square clear Perspex escape platform (11 × 11 cm) was submerged 0.5 cm below the water surface and placed at the midpoint of one of the four quadrants. The hidden platform remained in the same quadrant during training, while the start positions (N, S, E, or W) were randomized across trials. Mice were allowed up to 60 s to locate the escape platform, and their swimming paths were automatically recorded by the Noldus Ethovision system. On the last trial of the last training day, mice received a probe trial, during which the escape platform was removed from the tank and the swimming paths were recorded over 60 s while mice searched for the missing platform. The swimming paths were recorded and analyzed with the Noldus Ethovision system.\n\nOpto M3 multi-channel activity monitors (Columbus Instruments, OH, USA) were used to quantify spontaneous horizontal activity of animals. Monitors were placed in the colony area and testing was conducted in the same conditions of animal facility housing. All measurements were performed from 6:00 P.M. to 6:00 A.M. (dark phase) and to 6:00 A.M. to 6:00 P.M. (light phase), using animals maintained on a 12 hr light/dark cycle from 6:00 A.M. to 6:00 P.M. Individual mice were placed in 33 × 15 × 13-cm (length × width × height) clear plastic cages for 24h and total distance travelled was calculated from infrared beam breaks by determining activity at 1-min intervals. Horizontal activity was measured by the sequential breaking of infrared beams, 2.54 cm on center, in the horizontal plane of the x axis.\n\nAll statistical analyses were performed using SigmaStat Software. Differences between two groups were assessed with a two-tailed t test. The significance of factorial effects and differences among more than two groups were evaluated with ANOVA/RM ANOVA followed by Holm-Sidak test. Rank transformation was exploited for data not normally distributed. The level of significance was p < 0.05.\n\n\nResults\n\nIn order to determine the effectiveness of our approach for targeting CrT gene, the Cr levels were measured by GC/MS in various tissues. We observed a significant reduction of Cr in the brain (both cerebral cortex and hippocampus; Two Way ANOVA on ranks, post hoc Holm-Sidak method, p < 0.01 and p < 0.001 respectively), muscle (p < 0.01), heart (p < 0.001) and kidney (p < 0.05) of CrT−/y mice with respect to wild-type (WT) littermates (n = 4/tissue for each group; Table 1). To ensure that kidney Cr reduction was not due to impaired Cr biosynthesis, we also measured kidney production of guanidinoacetic acid (GAA). No difference was observed between CrT–/y (9.76 ± 0.71 nmol/mg of protein) and CrT+/y mice (10.70 ± 0.63 nmol/mg of protein; t test, p = 0.359).\n\nCr levels (mean ± SEM) in CrT–/y and CrT+/y animals (n = 4 per tissue for both groups). Cr levels have been measured by GC/MS. A reduction of Cr content was evident in the brain, muscle, heart and kidney tissue of mutant animals (Two Way ANOVA on ranks, post hoc Holm-Sidak method). * p < 0.05; p < 0.01; * p < 0.001.\n\nThe general appearance of CrT–/y mice was normal and no particular problems of breeding were observed. To evaluate the effects of CrT deletion on body weight, the mice with targeted disruption of CrT gene were weighed at P60, and compared with WT littermates. CrT−/y animals (n = 9) showed a significantly reduced body weight compared to CrT+/y animals (n = 9; t test, p < 0.01; Figure 2).\n\nAt P60 the weight of CrT–/y mice was significantly reduced compared to CrT+/y animals (CrT–/y: 18.75 ± 0.78 g, CrT+/y: 22.77 ± 0.90 g; t test, p < 0.01). , statistical significance. Error bars, s.e.m.\n\nWe first analyzed the general motor activity and anxiety-related behavior of CrT−/y (n = 9) and CrT+/y mice (n = 9) in the open field arena. Even though both groups of animals tended to avoid the center of the arena, remaining in the peripheral region for a significantly longer duration (Two Way ANOVA, post hoc Holm-Sidak method), the time spent by CrT−/y mutant mice in both the central and peripheral portion of the apparatus was not different from that recorded for WT animals (Two Way ANOVA, post hoc Holm-Sidak method, p = 0.725 and p = 0.922 respectively; Figure 3a, b, e). No difference between CrT−/y and CrT+/y animals was present even in motion speed and total distance moved (t test, p = 0.807 and p = 0.736 respectively; Figure 3c, d).\n\n(a, b) CrT–/y (n = 9) and CrT+/y mice (n = 9) spent a comparable amount of time in the center (CrT–/y: 75.16 ± 10.82 s, CrT+/y: 67.60 ± 18.11 s; a) and in the peripheral region (CrT–/y: 524.41 ± 10.87 s, CrT+/y: 526.52 ± 18.45 s; b) of the open field arena. A Two-Way ANOVA analysis shows no significant effect of genotype for both comparisons (p = 0.725 and p = 0.922, respectively). (c) Walking speed of animals during the exploration of open field arena. We found no significant difference (CrT–/y: 7.85 ± 0.43 cm/s, CrT+/y: 7.65 ± 0.71 cm/s; t test, p = 0.807). (d) The total distance moved in the open field arena did not differ between CrT mutants (4706.34 ± 258.75 cm) and WT animals (4535.28 ± 427.11 cm; t test, p = 0.736). (e) Representative examples of movement path during the open field session for a CrT–/y (left) and a CrT+/y mouse (right). Error bars, s.e.m.\n\nWe assessed declarative memory abilities in the object recognition test (ORT) evaluating animal ability to discriminate a new versus a familiar object. During the sample phase (Figure 4a), all experimental groups equally explored the objects, with a total exploration time of mutant mice (n = 8) very close to that recorded for the control group (n = 6; t test, p = 0.358). After a delay of 24h, the testing phase revealed that while CrT+/y mice displayed a clear preference toward the novel object spending a significantly longer time exploring it, an impaired performance was found in CrT–/y animals, which exhibited a significantly lower discrimination index than control animals (t test, p < 0.05, Figure 4b).\n\n(a) On the left, a schematic representation of the sample condition in object recognition task. Histograms depict the performance of CrT–/y and CrT+y during the sample phase: no difference in the total exploration time of objects was detected between the experimental groups (CrT–/y: n = 8, exploration time = 56.91 ± 5.40 s; CrT+/y : n = 6, exploration time = 67.20 ± 10.23 s; t test, p = 0.358). (b) On the left, a schematic diagram of the test condition. Histograms display object discrimination indexes of CrT–/y and CrT+y during the testing phase: a significantly lower discrimination index was found in CrT–/y mice (0.261 ± 0.053) compared to CrT+y animals (0.448 ± 0.059; t test, p < 0.05). , statistical significance. Error bars, s.e.m.\n\nTo evaluate whether CrT deletion may affect spatial working memory, we used the analysis of spontaneous alternation in the Y maze (Figure 5a). Animals of both groups equally explored all the three arms of the maze. Indeed, no effect of genotype was detected for either the number of entries in the single arms of the maze (designated A, B, C) or the total number of arm entries, indicating that the exploratory disposition of mutant animals (n = 9) was not altered compared to WT littermates (n = 9; Two-Way ANOVA, post hoc Holm-Sidak method, p = 0.640, p = 0.966, p = 0.252, p = 0.523 respectively, Figure 5b). In contrast, CrT−/y mice showed a significantly smaller rate of spontaneous alternation with respect to WT controls (t test, p < 0.05, Figure 5c).\n\n(a) Schematic diagram of the Y maze apparatus. (b) Histograms depict the mean number of entries in the single arms of the maze (A, B, C) and the total number of arm entries for the different experimental groups: animals of both groups equally explored all the three arms of the maze and general exploratory behavior of CrT–/y animals (n = 9; A: 15.22 ± 1.12, B: 14.22 ± 1.08, C: 12.22 ± 1.05, TOT: 41.67 ± 2.41) was totally comparable to that exhibited by WT littermates (n = 9; A: 14.00 ± 1.26, B: 14.11 ± 1.29, C: 15.22 ± 1.27, TOT: 43.33 ± 3.58; Two-Way ANOVA, post hoc Holm-Sidak method, p = 0.640, p = 0.966, p = 0.252, p = 0.523 respectively). (c) Alternation rate in the Y maze was significantly lower in CrT–/y mice (49.24 ± 3.20%) compared to that recorded for CrT+/y littermates (58.91 ± 2.99%; t test, p < 0.05). , statistical significance. Error bars, s.e.m.\n\nWe further assessed spatial memory abilities in the Morris water maze (MWM) task, a cognitive paradigm which allows testing both spatial learning and memory. Since a main effect of genotype was found on mean swimming speed recorded all along the training phase (t test, p < 0.05; Figure 6a), we analyzed path length, which is a quantity independent of swimming velocity. We found that the mean distance covered to locate the submerged platform on the last three days of training was longer in CrT–/y mice (n = 9) compared to CrT+/y littermates (n = 5; t test, p < 0.05; Figure 6b, c). To measure the strength of spatial learning and to discriminate between spatial and non-spatial memory strategies we performed a probe trial in which the hidden platform was removed and the amount of time spent in the former region of the platform was measured. The probe test confirmed the spatial memory impairment of CrT–/y mice: CrT+/y animals spent significantly longer time in the quadrant where the platform was located during the previous learning days (NE; Two-Way RM ANOVA, post hoc Holm-Sidak method, p < 0.05 for all comparisons); in contrast, CrT–/y mice showed no preference for the target quadrant, indicating that they did not remember the location of the hidden platform (Two-Way RM ANOVA, post hoc Holm-Sidak method; Figure 6d). A statistically significant effect of genotype was detected in the time spent exploring the target quadrant (Two-Way RM ANOVA, post hoc Holm-Sidak method, p < 0.05; Figure 6d).\n\n(a) Mean swimming speed measured all along the training phase for CrT–/y and CrT+/y animals: mutant mice (14.00 ± 0.53 cm/s) resulted to be slower swimmers with respect to control littermates (16.44 ± 0.60 cm/s; t test, p < 0.05). (b, c) Learning curves for CrT–/y (n = 9; blue) and CrT+/y mice (n = 5; grey) during the training phase. The histogram shows the mean swimming path covered to locate the submerged platform on the last three day of training for the two groups. A t-test analysis showed a statistical difference between CrT–/y (285.24 ± 37.53 cm) and CrT+/y animals (171.58 ± 23.80 cm; p < 0.05). (d) Probe trial. A Two-Way RM ANOVA followed by Holm-Sidak multiple comparison revealed that while CrT+/y spent significantly more time in the NE quadrant than in the other ones, CrT–/y did not show any preference for the target quadrant. In addition, the percentage of time spent in the target quadrant was shorter in CrT–/y mice (30.31 ± 5.33%) than in the other group (45.73 ± 7.35%). (e) Representative examples of swimming path during the probe session for a CrT–/y (left) and a CrT+/y mouse (right). , statistical significance. Error bars, s.e.m.\n\nTo investigate the presence of movement impairments in CrT−/y mice in a non-aversive environment, we investigated home-cage-locomotor activity. We found that CrT–/y mice (n = 9) are significantly less active than the CrT+/y group (n = 8, Two-Way ANOVA, post hoc Holm-Sidak method, p < 0.001). More specifically, CrT−/y mice showed decreased horizontal activity during the night period (Two-Way ANOVA, post hoc Holm-Sidak method, p < 0.001), while no effect of genotype was observed for exploration during daytime (p = 0.535; Figure 7a, b).\n\n(a) Total horizontal distance travelled throughout 24h (left), and over the dark (middle) or light phase (right). CrT–/y mice had a significant decrease in motor activity in comparison to control animals during the whole period of testing (CrT–/y: 43,594.22 ± 2,639.39 beam crossings, CrT+/y: 65,587.63 ± 5,831.19 beam crossings) and the night phase (CrT–/y: 29,109.67 ± 1,695.35 beam crossings, CrT+/y: 48,094.13 ± 4,843.56 beam crossings; Two-Way ANOVA, post hoc Holm-Sidak method, p < 0.001 for both comparisons), while the motor behavior of the two groups was similar in the day-time (CrT–/y: 14,484.56 ± 1,458.08 beam crossings, CrT+/y: 17,493.50 ± 2,957.57 beam crossings; p = 0.535). (b) Time course of horizontal activity of CrT–/y (blue) and CrT+/y (grey) animals during 24h. Data are plotted as total number of beam crossings ± SEM in each time block of 60 min. Dark and light phases are indicated. , statistical significance. Error bars, s.e.m.\n\n\nDiscussion\n\nWe have generated a new murine model of human CrT deficiency carrying a loss of function deletion of 5–7 exons in the murine orthologous of Slc68a gene. Given that most disease-underlying mutations in human CCDS1 lead to loss of CrT function (van de Kamp et al., 2014), our model has a good degree of construct validity. Beyond the genetic deletion, neurochemical abnormalities found in CrT−/y mice, reproducing the reduced levels of Cr that characterize the brain of CCDS1 patients (van de Kamp et al., 2012), are also helpful to confirm the successful disruption of CrT gene and the construct robustness of this model. Importantly, Cr deficiency is apparent in both the cerebral cortex and hippocampus, i.e., two brain regions crucially involved in the patient cognitive defects. These results seem to support the hypothesis that, despite AGAT and GAMT expression (Carducci et al., 2012; Schmidt et al., 2004; Tachikawa et al., 2004), in CrT deficiency conditions endogenous synthesis does not compensate for the loss of Cr uptake in the mouse (Skelton et al., 2011) as in the human brain (Cecil et al., 2001). In contrast to the preservation of Cr levels in skeletal muscle of CCDS1 patients (deGrauw et al., 2003), we observed that mutant mice exhibit Cr reductions in muscle and other peripheral tissues. This observation is in agreement with data showing that skeletal muscle tissue from a different CrT knockout mouse displayed a dramatic reduction of Cr levels (Russell et al., 2014; Skelton et al., 2011).\n\nOur behavioral investigation highlighted that CrT−/y mice carrying a different deletion than previously reported (Skelton et al., 2011) exhibit a broad spectrum of phenotypes establishing the validity of this model and corroborating its utility in translational studies. Mutant mice, indeed, show cognitive impairments in a battery of learning and memory tests aimed at assessing both explicit and implicit memories such as object-recognition task, Y maze and Morris water maze. The memory deficiency assessed across a variety of behavioral tasks indicates that CrT−/y animals have a general cognitive impairment, which is a key clinical feature in CCDS1 patients.\n\nWhile the motor development is only mildly delayed in CCDS1 patients (van de Kamp et al., 2013) and myopathic symptoms have been rarely described (Anselm et al., 2006; van de Kamp et al., 2013), mostly as late onset deficits (deGrauw et al., 2002; Hahn et al., 2002; Kleefstra et al., 2005), we found that reduced muscle levels of Cr measured in mutant animals were accompanied by alterations of motor behavior. CrT−/y mice, indeed, showed significantly decreased home-cage-locomotor activity (particularly evident during the night period) and they were slower swimmers than CrT+/y mice. In contrast, we found that vulnerability to stress and anxiety responses are not sensitive to CrT deletion. Future studies using conditional mouse models with a disruption of CrT allele only in the brain tissue will be useful to dissect the role of peripheral Cr in the development of cognitive deficits. It has been reported that a CrT deletion exclusively restricted to forebrain excitatory neurons during late postnatal development induces selective learning and memory deficits without affecting motor behavior (Kurosawa et al., 2012).\n\nBecause of the importance of Cr in normal retinal function and development (Acosta et al., 2005), it has been suggested that an alteration of visual capabilities might play a role in the cognitive deficits displayed by CrT−/y animals. We reported that during the ORT sample phase all experimental groups equally explored and observed the objects, with the total exploration time of mutant mice very close to that recorded for the control group, suggesting that the visual system is not impaired in CrT–/y animals. In addition, to avoid possible confounding effects due to reduced visual acuity, the tank used in the Morris water maze task was surrounded by a set of extra-maze cues in a visual discrimination range detectable even by partially-sighted animals.\n\nIn conclusion, this CrT−/y murine model will provide a new tool for improving preclinical evaluation of potential CCDS1 intervention treatments. The results confirm previous data suggesting that CCDS1 can be well modeled in mice (Kurosawa et al., 2012; Skelton et al., 2011). Null mice display an impairment of motor behavior rarely present in human patients; however, the use of conditional mice will avoid this problem. Since CCDS1 is still an untreatable pathology, there is a compelling need for developing effective therapeutic strategies. The availability of murine models that reliably reproduce the human condition will fuel and support the research in this field. To assess the reproducibility and the predictive validity of promising treatments for CCDS1 as well as for other disorders, the validation of findings in more than one animal model is strongly desirable prior to launching later-stage translational or clinical projects (Katz et al., 2012). Since another invalidating hallmark of CCDS1 is the frequent occurrence of seizures, additional studies in CrT−/y mice analyzing the behavioral response to kainic-acid injection will be required to provide useful information about seizure susceptibility in this model.\n\n\nData availability\n\nF1000Research: Dataset 1. Data for neurochemical and behavioral assessment in a mouse model of creatine deficiency, 10.5256/f1000research.5369.d36153 (Baroncelli et al., 2014).", "appendix": "Author contributions\n\n\n\nGC, VL and TP conceived the study. TP designed the experiments. LB, MGA, JT, EP and MM carried out the research. EA and CG provided the mouse model. LB and TP wrote the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nAcosta ML, Kalloniatis M, Christie DL: Creatine transporter localization in developing and adult retina: importance of creatine to retinal function. Am J Physiol Cell Physiol. 2005; 289(4): C1015–1023. PubMed Abstract \| Publisher Full Text\n\nAlessandri MG, Celati L, Battini R, et al.: Gas chromatography/mass spectrometry assay for arginine: glycine-amidinotransferase deficiency. Anal Biochem. 2005; 343(2): 356–358. PubMed Abstract \| Publisher Full Text\n\nAnselm IA, Alkuraya FS, Salomons GS, et al.: X-linked creatine transporter defect: a report on two unrelated boys with a severe clinical phenotype. J Inherit Metab Dis. 2006; 29(1): 214–219. 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PubMed Abstract \| Publisher Full Text\n\nWyss M, Kaddurah-Daouk R: Creatine and creatinine metabolism. Physiol Rev. 2000; 80(3): 1107–1213. PubMed Abstract" }	[ { "id": "6247", "date": "02 Oct 2014", "name": "Benedetto Sacchetti", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the present paper the authors describe a new murine model of CCDS1 obtained by ubiquitous deletion of 5-7 exons in the Slc6a8 gene. The experiments in general are well controlled and the results could be of interest for a general audience. However, I think there are two related points that should be added or clarified before the paper can be considered for publication. The authors reported that Cr depletion altered spontaneous locomotor behavior (Fig 7) and reduced muscle levels. How can this data fit with the normal behavior (namely, the motion speed and the total distance moved) displayed by mutant animals in the open field test? On the other hands, can the authors exclude that the aforementioned reduced motor activity may have interfere with learning and memory trials? The authors should at least discuss this possibility.", "responses": [ { "c_id": "1065", "date": "06 Nov 2014", "name": "Laura Baroncelli", "role": "Author Response", "response": "We agree that the difference between the altered spontaneous locomotor behavior of mutant animals in the home cage and the normal exploratory disposition in the open field arena can be a little bit surprising. However, we feel that the lack of a genotype effect for the latter measure may be due to the aversive nature of the open field arena, which may affect the explorative behavior of both wild-type and mutant mice, thus masking the difference in motor activity between the two groups. We think that our data allow to exclude the possibility that an impaired motor activity can interfere with the presented results of learning and memory test. We found that, during the ORT sample phase, the total exploration time of objects was equal for mutant and control mice (Figure 4a), and animals of both groups equally explored the Y maze in terms of both the number of entries in the single arms of the maze and the total number of arm entries (Figure 5b). These results strongly suggest that animals’ level of activity does not affect their cognitive performance. As for the Morris water maze during the training phase we analyzed the path length covered to locate the submerged platform just to avoid the confounding effects of the reduced swimming speed observed in mutant mice that should not affect instead the performance in the probe trial." } ] }, { "id": "6560", "date": "05 Nov 2014", "name": "Luis M. Valor", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn “A novel mouse of creatine transporter deficiency” the authors describe the phenotype of a new knockout mouse for Slc6a8 gene which is associated with a depletion in the levels of creatine in diverse organs. This phenotype is reminiscent of the CCDS1 symptomatology, therefore the main output of the present report is an increase in the number of available murine models for this disorder as claimed in the article. The paper is well written and the work is well presented, with no major concerns regarding the data as shown. Nonetheless, I miss a more complete behavioural analysis. In some cases this is not crucial because assessment of particular tasks is expected to support current findings (absence of anxiety in the open field or impaired spatial working memory in the Y-maze) although with the strength of using more dedicated paradigms (elevated plus maze or T-maze based on a rewarding system, respectively). However, it is more relevant in the case of motor and neuromuscular deficits to enhance the conclusions obtained from spontaneous activity measurements, and other approaches (accelerating rotarod, grip strength, vertical pole, etc. to put some examples) may be more informative.", "responses": [ { "c_id": "1181", "date": "22 Jan 2015", "name": "Laura Baroncelli", "role": "Author Response", "response": "We agree that a more detailed behavioral analysis (in particular dedicated to the understanding of motor and neuromuscolar deficits) would be informative for the characterization of CCDS1 murine models. We plan to do this in our next paper." } ] }, { "id": "6559", "date": "11 Nov 2014", "name": "Andreas Schulze", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe research group generated a new ubiquitous CrT knockout as mouse model for creatine transporter deficiency with a large 3 exon deletion in the Slc6a8 gene. Biochemical phenotyping revealed creatine deficiency in brain, muscle, heart, and kidneys and behavioral testing revealed a phenotypic similarities with CrT patients. Therefore the new mouse model appears to be a valid tool to study creatine transporter deficiency. What needs some more elaboration is the discrepancy in findings compared to the mouse model described by Skelton et al. (2011). Considering the similarities of the knock-outs, both have a large deletion of three exons, on would expect similar findings. But in the knock-out presented here there is more cognitive impairment, i.e. novel object recognition was abnormal while it was normal in the Skelton paper, and this is despite of the fact that the brain creatine deficiency reported here appears to be less pronounced than in the mouse model of Skelton et al. Before indexing the authors should provide information on whether the mouse chow contained creatine (some mouse chow contains fish meal and the latter contains creatine). Also it would be important to know the creatine concentration in plasma. The creatine concentration in mutants is expected to be higher than in wild types. I wonder whether blood contamination has contributed to unexpected high brain creatine concentration in mutants. Why did the group not consider whole-body perfusion prior to organ removal? Did the authors measure creatine/creatinine ratios in urine? And why not providing the information on guanidinoacetate in organs and body fluids as well?", "responses": [ { "c_id": "1127", "date": "16 Dec 2014", "name": "Skelton Lab", "role": "Reader Comment", "response": "Dr. Schulze,I would like to respectfully clarify an error in your review. Our ubiquitous CrT mice did indeed show deficits in object recognition memory, as shown in figure 6 of our PLOS One paper. In fact, the object recognition deficit was similar to the deficit presented in this paper. Best Regards,Matthew R. Skelton, Ph.D.Cincinnati Children's Research Foundationmatthew.skelton@cchmc.org" }, { "c_id": "1182", "date": "22 Jan 2015", "name": "Laura Baroncelli", "role": "Author Response", "response": "As already clarified by Dr. Skelton, our behavioral findings are not at odds with those reported in his PLOS ONE paper: indeed, their knockout mice show a deficit in object recognition memory very similar to that measured in our model. The difference in the extent of creatine deficiency is explained by the different methods employed to measure this metabolite: in our study creatine was analyzed using gas chromatography/mass spectrometry, a widely accepted specific and sensitive technique, whereas Skelton et al. (2011) used a less sensitive colorimetric method. As regards the mouse chow, the manufacturer told us that the pellet purchased for our animal facility is not added with creatine. We made this point clearer, adding a sentence in the Materials and methods section (Animal housing subsection). We followed your suggestion and measured creatine concentration also in body fluids, and more specifically in serum and urine. We observed a significant reduction of Cr in the serum of CrT−/y mice with respect to wild-type (WT) littermates (Two Way ANOVA on rank transformed data, post hoc Holm-Sidak method, p < 0.001). In contrast, an increase of Cr levels (Two Way ANOVA on rank transformed data, post hoc Holm-Sidak method, p < 0.05) and creatine/ creatinine ratio was present in the urine of mutant with respect to WT animals (t test, p < 0.001). We added these data in the Result section and in Table 1. We also modified the discussion adding the following sentences: “In contrast to the preservation of Cr levels in skeletal muscle of CCDS1 patients (deGrauw et al., 2003), we observed that mutant mice exhibit Cr reductions in muscle and other peripheral tissues and body fluids. This observation, which is in agreement with data from a different CrT knockout mouse (Russell et al., 2014; Skelton et al., 2011), further confirmed that the recombination resulted in a ubiquitous disruption of the CrT gene. In particular, the reduction of serum Cr level may be explained by defective gut absorption from the diet (Garcia-Miranda et al., 2009; Skelton et al., 2011). The only body fluid in which Cr levels resulted to be increased is urine; it is likely that the lack of a functional transporter impairs the creatine salvage normally operated by the kidney (van de Kamp et al., 2014). Consistently, we found an elevated creatine/creatinine (Cr/Crn) ratio in the urine of mutant mice, probably due to a combination of reduced renal reabsorption of creatine and decreased creatinine excretion.” Since we found a strong reduction of creatine concentration, we don’t think that blood contamination could be responsible for higher brain Cr levels in these mice. Finally, we provided the information on GAA content in organs and body fluids, adding a Table 2 to the manuscript." } ] } ]	1	https://f1000research.com/articles/3-228
https://f1000research.com/articles/4-20/v1	22 Jan 15	{ "type": "Research Note", "title": "Advantages of distributed and parallel algorithms that leverage Cloud Computing platforms for large-scale genome assembly.", "authors": [ "Priti Kumari", "Raja Mazumder", "Vahan Simonyan", "Konstantinos Krampis", "Priti Kumari", "Raja Mazumder", "Vahan Simonyan" ], "abstract": "Background: The transition to Next Generation sequencing (NGS) sequencing technologies has had numerous applications in Plant, Microbial and Human genomics during the past decade. However, NGS sequencing trades high read throughput for shorter read length, increasing the difficulty for genome assembly. This research presents a comparison of traditional versus Cloud computing-based genome assembly software, using as examples the Velvet and Contrail assemblers and reads from the genome sequence of the zebrafish (Danio rerio) model organism.Results: The first phase of the analysis involved a subset of the zebrafish data set (2X coverage) and best results were obtained using K-mer size of 65, while it was observed that Velvet takes less time than Contrail to complete the assembly. In the next phase, genome assembly was attempted using the full dataset of read coverage 192x and while Velvet failed to complete on a 256GB memory compute server, Contrail completed but required 240hours of computation.Conclusion: This research concludes that for deciding on which assembler software to use, the size of the dataset and available computing hardware should be taken into consideration. For a relatively small sequencing dataset, such as microbial or small eukaryotic genome, the Velvet assembler is a good option. However, for larger datasets Velvet requires large-memory compute servers in the order of 1000GB or more. On the other hand, Contrail is implemented using Hadoop, which performs the assembly in parallel across nodes of a compute cluster. Furthermore, Hadoop clusters can be rented on-demand from Cloud computing providers, and therefore Contrail can provide a simple and cost effective way for genome assembly of data generated at laboratories that lack the infrastructure or funds to build their own clusters.", "keywords": [ "Next Generation sequencing", "genome assembly", "Cloud Computing" ], "content": "Background\n\nThe earliest landmark in genome sequencing is that of the Bacteriophage MS21 between 1972 and 1976. Following that, two DNA sequencing techniques for longer DNA molecules were invented, first the Maxam-Gilbert2 (chemical cleavage) method and then the Sanger3 (or dideoxy) method. The Maxam-Gilbert method was based on nucleotide-specific cleavage by chemicals, and is best applied to oligonucleotides that and short sequences usually smaller than 50 base-pairs in length. On the other hand, the Sanger method was more widely used because it leveraged the Polymerase Chain Reaction (PCR) for automation of the technique, which also allows to sequence long strands of DNA including entire genes. In more detail, the Sanger technique is based on chain termination by dideoxynucleotides triphosphate (ddNTPs) during PCR elongation reactions (review in4). Although the automated Sanger method had dominated the industry for almost two decades, with sequencing applications and broad demand for the technology in genome variation studies, comparative genomics, evolution, forensics, diagnostic and applied therapeutics, it was still limiting due to its high cost and labor intensive process5.\n\nThe high demand for low-cost sequencing led to the development of high-throughput technologies also known as Next Generation Sequencing (NGS), that parallelize the sequencing process and lower the cost per sequenced DNA base-pair. NGS techniques achieve this by automating template preparation and using high-speed, precision fluorescence imaging, for highly parallel identification of the nucleotides. NGS technologies involve sequencing of a dense array of DNA fragments by iterative cycles of enzymatic manipulation and imaging-based data collection. The major NGS platforms are Roche/454FLX (http://454.com/), Illumina/Solexa Genome Analyzer (http://www.illumina.com/systems/genome_analyzer_iix.ilmn), Applied Biosystems SOLiD system (http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-generation-sequencing.html), Helicos Heliscope (http://www.helicosbio.com/Products/HelicosregGeneticAnalysisSystem/HeliScopetradeSequencer/tabid/87/Default.aspx) and Paciﬁc Biosciences (http://www.pacificbiosciences.com/). Summary statistics for the throughput and characteristics of these NGS platforms are shown Figure 1, while additional review studies are available in the literature6–8.\n\nAlthough these platforms are quite diverse in sequencing biochemistry as well as in how the array is generated, their workflows are conceptually similar. First, a library is prepared by fragmenting PCR-amplified DNA randomly, followed by in vitro ligation to a common adaptor, that is small DNA oligonucleotide with known sequence. In the next step, clustered amplicons that are multiple copies of a single fragment are generated and serve as the sequencing fragments. This can be achieved by several approaches, including in situ polonies9, emulsion10 or bridge PCR11,12. Common to these methods is that PCR amplicons derived from any given single library molecule end up spatially clustered, either to a single location on a planar substrate (in situ polonies, bridge PCR), or to the surface of micron-scale beads, which can be recovered and arrayed (emulsion PCR).\n\nThe advantage of the NGS platforms is sequencing using single, ampliﬁed DNA fragments, avoiding the need for cloning required by the Sanger method. This also makes the technology applicable to genomes of un-cultivated microorganism populations, such as for example in metagenomics. A disadvantage of the new technology is that sequence data is in the form of short reads, presenting a challenge to developers of software and genome assembly algorithms. More specifically, it can be difficult to correctly assign reads and separate between genomic regions that contain sequence repeats even if using high-stringency alignments, especially if the lengths of the repeats are longer than the reads. In addition, repeat resolution and read alignments are further complicated by sequencing error, and therefore genome assembly software must tolerate imperfect sequence alignments. A reduced alignment stringency can return many false positives that results in chimeric assemblies where distant regions of the genome are mistakenly assembled together. The limitation of short reads is compensated by multiple overlaps of the sequenced DNA fragments, resulting in many times coverage of the genome sequence. Finally, genome assembly is hindered by regions that have nucleotide composition for which PCR does not achieve its optimum yield, resulting in non-uniform genome coverage that in turn leads to gaps in the assembly.\n\nTo alleviate some of these problems in genome assemblies with short reads, “paired-end” reads are used that are generated by a simple modification to the standard sequencing template preparation, in order to get the forward and reverse strands at the two ends of a DNA fragment. The unique paired-end sequencing protocol allows the user to choose the length of the insert (200–500 bp) and use the positional and distance information of the reads for validating the genome assembly, allowing for resolution of alignment ambiguities and chimeric assemblies.\n\nTwo approaches widely used in NGS genome assemblers are the Overlap/Layout/Consensus (OLC)13 and the de Bruijn Graph (DBG)14 method, both using K-mers as the basis for read alignment. A graph is an abstraction used in computer science, and is composed of “nodes” connected by “edges”. If the edges connecting the nodes can be traversed in one direction, the graph is a directed graph, whereas if the edges can be traversed in both the directions the graph is bi-directed15.\n\nThe OLC approach for genome assembly is the typical method for Sanger datasets, and is implemented in software such as Celera Assembler, PCAP and ARACHNE16–18. With this approach reads are represented as nodes in the graph, and nodes for overlapping reads are connected by an edge. In more detail, OLC assembles proceed in three phases: in the first phase overlap discovery involves all-against-all comparison, pair-wise read alignment. The algorithm used for that purpose is a seed and extend heuristic algorithm that pre-computes K-mers from all reads, then selects overlap candidate reads that share K-mers and calculates alignments using the K-mers as alignment seeds. This step of the algorithm is sensitive to settings of K-mer size, minimum overlap length and percent identity required to retain an overlap as true positive, in addition to base calling errors and low-coverage sequencing. Using the results from the read alignment, an overlap graph is constructed that provides an approximate read layout. The overlap graph does not include the sequence base calls rather than just the overlapping read identifiers, so large-genome graphs may fit into practical amounts of computer memory. The graph also has metadata on the nodes and edges, in order to distinguish the lengths, 5′ and 3′ ends, forward and reverse complements, and the type of overlap between the reads. In the second phase, the precise layout and the consensus sequence of the graph are determined by performing Multiple Sequence Alignment (MSA)19. For that purpose, the algorithm must load all the sequences into memory, and this becomes one of the most computationally demanding stages of the assembly. Finally, in the third phase the assembly algorithm follows a Hamiltonian path20 to “walk” through the graph visiting every node only once, and the contigs formed by merging the overlapping reads are determined.\n\nThe second approach in genome assembly is based on the de Brujn Graph (DBG) algorithms, and example software using this approach includes Velvet, Contrail, ALLPATHS and SOAPdenovo21–24. In the DBG approach each node in the graph corresponds to a unique K-mer present in the set of reads, and a directed edge connects two nodes labeled ‘a’ and ‘b’, if the k – 1 length suffix of ‘a’ is the same as the k – 1 length prefix of ‘b’. For example, a graph node representing the K-mer (K=3) ATG will have an edge with the node representing TGG. The DBG algorithms are more efficient for large datasets, as they doe not require all-against-all read alignment and overlap discovery, while it also doe not store individual reads or their overlaps in the computer memory. A de Brujn graph can either be uni-directed or bi- directed, with a single edge connecting two nodes, or edges with two directions for the 5’ or 3’ genome strands. A bidirected graph has the advantage of allowing simultaneous assembly of sequence reads from both strands of the genome15. The DBG assembly algorithms traverse the graph using an Eularian path14, where each edge is visited exactly once.\n\n\nMethods\n\nAssembly dataset: The sequence reads dataset were Illumina reads from the fish species M. zebrafish downloaded from Assemblathon database (http://assemblathon.org). The genome was approximately 1GB in size, the total library coverage estimate for this dataset was 197X. The read length for the given dataset was 101 and in the first phase 38,364,464 reads with coverage of only 2X, both paired and unpaired, totaling 2,762,241,408 base pairs were used (filtered for bad quality reads). Following that, different variations in the size of dataset used for running the assembly involving 1/4, 1/2 and the entire dataset of fish genome. The dataset consisted of pairs of files with each corresponding to the paired-end reads. De novo assembly was performed using two different assemblers, Velvet and Contrail, both using the graph-theoretic framework of De-Brujn Graph. Velvet requires high performance computer servers with large RAM memory, whereas Contrail is a distributed memory assembler that performs the computation in parallel over several servers on a computer cluster.\n\nAssembly statistics: The output of the assembly was a file with a list of contigs of various lengths. The file contains contigs along with various details like length and coverage. For the Velvet assembler, the output file was parsed using a Perl script called velvet_stats.pl to calculate various assembly statistics. Similarly, for the Contrail assembler a Perl script called contrail_stats.pl was used. These scripts are available upon request from the authors. Using the Perl scripts the following statistics were calculated for comparing the quality of the two assemblies:\n\n1) N50 score: The length of the largest contig for which the following is true: the sum of its length and the lengths of all larger contigs equals to 50% of the total contig length. The N50 size is computed by sorting all contigs from largest to smallest and by determining the minimum set of contigs whose sizes total 50% of the entire genome.\n\n2) Maximum contig length: Contig with largest number basepairs.\n\n3) Minimum contig length: Contig with smallest number of basepairs.\n\n4) Mean contig length: Average of all the contigs length.\n\nTo the run the assembly on the full dataset using contrail, all the read files were merged into a single file, and copied in a Hadoop compute cluster available at JCVI that consisted of 8 servers with 32 GB RAM and 16cores each.\n\nBoth the Velvet and Contrail assemblers are designed for short reads. Velvet requires a compute server with large RAM memory, whereas Contrail relies on Hadoop to distribute the assembly graphs across multiple servers. Velvet is a de novo genomic assembler specially designed for short read sequencing technologies, such as Solexa or 454. It currently takes in short read sequences, removes errors then produces high quality, unique contigs using paired-end read and long read information when available, for resolving genomic repeats that complicate contig calculation. Velvet resolves errors which can arise due to both the sequencing process or to the polymorphisms by removing the “tips” in the de Bruijn graph that are chain of nodes that is disconnected on one end, or “bubbles” using the Tour Bus algorithm, both originating from nucleotide differences due to sequencing error or polymorphisms in diploid genomes.\n\nContrail enables de novo assembly of large genomes from short reads by leveraging Hadoop, a software library and framework that allows distributed processing of large data sets across clusters of computers using a simple programming model. The Hadoop Distributed File System (HDFS) is the primary storage system in this framework, and creates multiple data blocks distributed across compute server throughout a cluster to enable reliable, extremely rapid parallel computations. HDFS is highly fault-tolerant and is designed to be deployed on low-cost, commodity hardware. Build on top of HDFS is the Hadoop-MapReduce Framework that uses a “Map” step in which individual data records tagged with a “key” are processed in parallel. In a second step, a “Reduce” function performs aggregation and summarization, in which all associated records based on the key are brought together from across the nodes of the cluster.\n\nMore specifically, the Hadoop implementation of the Contrail algorithm in the Map phase scans each read and emits the key-value pairs (u, v) corresponding to overlapping k-mer pairs that form an edge. After the map function completes, a key sorting phase that is executed in parallel groups together edges for the same K-mer based on the key value. The initial graph construction creates a graph with nodes for every K-mer in the read set. This is followed by aggregation of identical K-mers in the Reduce phase, where also linear paths of the de Bruijn graph are calculated and continuously overlapping K-mers are simplified into single graph nodes representing longer stretches of sequence.\n\nContrail Assembly Steps: The Contrail source code was downloaded from sourceforge.net repository (http://sourceforge.net/apps/mediawiki/contrail-bio/index.php?title=Contrail). Since Contrail uses Hadoop MapReduce programming framework, the reads file from the fish dataset were stored in the Hadoop File System (HDFS), on a local Hadoop cluster with 5TB storage capacity distributed across 8 nodes, with each node having 32GB RAM and 16 cores memory. In order to run the assembly the following steps were followed:\n\n1) Reads files were downloaded from Assemblathon database using wget http://bioshare.bioinformatics.ucdavis.edu/Data/hcbxz0i7kg/Fish/62F6HAAXX.1.1.fastq.gz\n\n2) Copy the unzipped fastq files into the Hadoop File System (HDFS) using ‘put’ command: /opt/hadoop/bin/hadoop/fs –put/source folder/destination folder in HDFS. ‘Put’ command copies files from the local file system to the destination HDFS, also splitting and distributing the file equally across several compute nodes on the cluster.\n\n3) From within the Hadoop Server enter the directory storing the source code for contrail assembler.\n\n4) Run the assembly using the command: contrail.pl –reads [readfile_path] –hadoop [destination folder] –start [stage_of_assembly] [K-mer length].\n\n5) From the output directory obtain the statistic folder, final graph folder and the final contig folder (fasta format) using the Hadoop ‘get’ command. ‘Get’ command copy files to the local file system from HDFS.\n\n6) Extract the files in the final graph folder using cat and gunzip command and store all the graph in a single file: cat part* \| gunzip >final_graph.\n\n7) Parse the final graph with the perl script contrail_stats.pl and save the output in a file.\n\nVelvet Assembly Steps: The Velvet assembler was downloaded from the EBI website (http://www.ebi.ac.uk/~zerbino/velvet/). The first component of the assembler is Velveth takes in a number of sequence files, produces a hash table, and then outputs two files in an output directory, “Sequences” and “Roadmaps”, which are necessary for the next step of the assembler called Velvetg. Users need to provide the output directory folder, file format of the sequence stored in the read file, hash length or K-mer length, read type, and read file name. The second Velvetg component of the assembler is where de Brujn graph is built. It has various options like specifying coverage cutoff or minimum length of the contigs to output. Velvet has optional parameters that allow to assemble paired reads data as well. To activate the use of read pairs, two parameters must be specified: the expected (i.e. average) insert length (or at least a rough estimate), and the expected short-read coverage. The insert length is understood to be the length of the sequenced fragment, i.e. it includes the length of the reads themselves.\n\n1) Run Velveth on one fourth of the entire dataset using unpaired reads using the command velveth/outputdir 65 –fastq –short read_file_1 read_file_2.\n\n2) Run Velvetg on the output obtained from Step 1. using velvetg/outputdir.\n\n3) Run the Velvet on paired read files using velveth/outputdir 65 –fastq –shortPaired read_file_1 read_file_2.\n\n4) Run velvetg on the output obtained from Step 3 using velvetg output_directory/-ins_length 101 -exp_cov 20.\n\n5) After the assembly is complete, parse the stats.txt file from the output directory using the Perl program velvet_stats.pl.\n\n\nResults and discussion\n\nThe comparison of the de novo assembly quality for the Velvet and Contrail algorithms presented in the current study, is based on the size of the dataset and assembly statistics using both paired and un-paired sequence data. In the first phase of the research, we compared assembly results for a range of K-mers, using a minimal portion of zebrafish data set (single lane un-paired, approximately 2X coverage). For this dataset, Figure 2 shows the graph plot with the relationship between the K-mer value and the maximum contig size in the assembly output for both Velvet and Contrail assemblers. The graph indicates that the maximum contig length increases with increase in the K-mer size, but above K-mer value of 65 that is a little more than half of the actual read length (101 for our dataset), the contig size decreases indicating lower assembly quality. The reason is that at larger K-mer lengths, the DBG algorithm connects two K-mers with an edge on the assembly graph only if K-1 length suffix of the first K-mer is same as K-1 length prefix of the second K-mer. As the K-mer approaches the actual read size, the probability that any pair of K-mers will have a K-1 length of suffix/prefix identity, decreases significantly given also the presence of sequencing errors. From Figure 2 it is also apparent that for both assemblers the maximum contig size was achieved at K-mer size 65, and therefore the remaining experiments in this study were carried out using that specific value.\n\nIn the next step, assembly was performed with Velvet for the same 2X coverage dataset using paired reads, but no significant difference was observed in the maximum contig size (Table 1, row 1–2). Paired read assembly was not performed for Contrail, as the algorithm implementation used in the current study did not provide this feature. The un-paired datasets with Contrail returned half the maximum contig size of Velvet (Table 1, row 3), and took significantly more computing time to complete due to the overhead required for initiating the parallel computation with the Hadoop cluster (see Methods section for details).\n\nIn the second phase of the research we attempted to perform assembly using the entire dataset of the zebrafish genome that has approximate coverage of 192X. For running the assembly using Velvet, first the sub-command Velveth was used that creates a “roadmap” of read overlaps, followed subsequently by Velvetg to create and traverse the de Bruijn graph. While we used a 1 Terabyte RAM memory server to run the assembler and the Velveth step of the assembly completed, Velvetg failed to complete, and similar situation was observed when using paired reads (Table 1, row 4–5). The error reported by the Velvet software in both cases indicated that there was not sufficient memory for the completion of the assembly computation. On the other hand, the Contrail assembler successfully completed the run and returned the best N50 score and largest contig size in the current study, albeit requiring 240 hours running time (Table 1, row 6).\n\nSince Velvet failed to complete the assembly for the entire genome, a partial set of the reads from the zebrafish dataset was used as input. First, by using one-fourth of the total dataset the assembly completed in nine and twelve hours for un-paired and paired reads respectively (Table 1, row 7–8). In this case, there was significant difference in the assembly quality between paired and unpaired reads, with the maximum contig size for unpaired reads approximately 7,000bp shorter when compared to paired reads. Next, one-half of the zebrafish dataset was used as input to Velvet, and with unpaired reads the total assembly time completed successfully in 24 hours (Table 1, row 9). In detail, the Velveth step consumed approximately 8hours, while Velvetg required an additional 14 hours. The maximum contig size obtained was about half of that with the Contrail assembler when using the full dataset of unpaired reads (Table 1, row 6). In addition the assembly for Velvet with one-half of the unpaired reads dataset, was found to be comparable to the Velvet results using one-fourth of the paired reads (Table 1, row 8–9). This demonstrates that is feasible to achieve better assembly with less data, when paired end reads are available. Finally, using half of dataset with paired reads, the Velveth step completed in 10hrs while Velvetg failed on our 1TB compute server due to insufficient memory space (Table 1, row 10).\n\n\nConclusions\n\nThe present study presents an example comparison of performance characteristics for distributed genome assemblers that leverage parallel computing and commodity, cloud computing clusters, versus assemblers that require large memory, specialized and expensive compute servers. The overall conclusion is that for assembling small datasets, reads from bacterial genomes or small eukaryotic genomes, it is better to use assembly software such as Velvet. Nonetheless, for larger genomes and datasets a single server cannot scale, and the assembler fails to load the entire graph into the memory and the assembly does not complete. Genome assemblers such as Contrail present an alternative option, as it performs assembly in parallel using cloud computing and commodity server clusters. Contrail is based on the Hadoop programming framework that is open source, freely available and also can be installed on local cluster. If a local clusters are not available, Hadoop compute servers can be rented from cloud providers. Depending on the size of the assembly to be carried additional nodes can be attached or rented as needed, and expand the capacity of the Hadoop cluster on the cloud or locally. Therefore, additional research by the bioinformatics community on cloud-based, scalable assemblers can result in cost effective solutions for assembly of genomes of any size for both well established institutions or smaller, academic research groups.", "appendix": "Author contributions\n\n\n\nP.K. performed the research and wrote the manuscript, R.M. and V.S. contributed to the research and writing the manuscript, K.K. reviewed and edited the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis project has been funded in whole or part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services under contract numbers N01-AI30071 and/or HHSN272200900007C.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nMin Jou W, Haegeman G, Ysebaert M, et al.: Nucleotide sequence of the gene coding for the bacteriophage MS2 coat protein. 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Publisher Full Text\n\nZerbino DR, Birney E: Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 2008; 18(5): 821–829. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchatz MC, Langmead B, Salzberg SL: Cloud computing and the DNA data race. Nat Biotechnol. 2010; 28(7): 691–3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nButler J, MacCallum I, Kleber M, et al.: ALLPATHS: de novo assembly of whole-genome shotgun microreads. Genome Res 2008; 18(5): 810–820. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi Y, Hu Y, Bolund L, et al.: State of the art de novo assembly of human genomes from massively parallel sequencing data. Hum Genomics. 2010; 4(4): 271–7. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "7605", "date": "10 Feb 2015", "name": "Keith E. Robison", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis title of this manuscript would lead a reader to believe that a careful comparison of two broad strategies for de novo sequence assembly. Unfortunately, what the manuscript delivers is an error-rich and outdated introduction, incompletely defined methods and an extremely limited comparison on a single dataset of two assembly programs. In their introduction the authors dig far into the history of DNA sequencing nearly to the very beginning. However, this summary is filled with dubious assertions that lack citations. For example, they credit PCR with boosting Sanger sequencing over Maxam-Gilbert sequencing, but Sanger sequencing had already all-but-extinguished Maxam-Gilbert before PCR had become commonly used in any facet of sequencing, and even today Sanger sequencing does not have a reliance on PCR (the authors may be confusing cycle sequencing, which relies on a linear amplfication using thermostable polymerases, with PCR). The authors present in figure form a comparison of four \"next generation sequencing\" systems (a term that really should be retired, given the fact that these systems are over a decade old now). The figure is exquisitely badly formatted and nearly unreadable when printed due to using a thin white font on dark backgrounds; if the information was of any value it should have been formatted as a table. Alas, the information in the table is worse than its formatting, being completely out-of-date.The statistics given for Illumina sequencing, which name an instrument (the GA) discontinued several years ago, give a cost per base that is roughly right for the MiSeq platform, but the read lengths on that platform are far longer (now 2x300). Several of the other Illumina platforms offer longer read lengths than given with a cost per basepair which are several orders of magnitude lower than given in the figure.Another quadrant of the figure describes the SOLiD system, which has rarely been used for de novo assembly. In any case, the number of reads per run and read length are both wrong, which leads to the cost per basepair being off by over an order of magnitude.A third quadrant gives obsolete statistics for the 454 platform, which hit read lengths of over 800 bases (or >2X that given in the figure). However, that really doesn't matter except historically since Roche discontinued the 454 platform in 2014. Even worse is the 4th quadrant, which describes the Helicos sequencer, a comparny that went bankrupt in 2011.The Ion Torrent systems, despite being used frequently for small genome de novo assembly, are not mentioned anywhere. Missing from the table, but briefly mentioned in the text, is the Pacific Biosciences platform. Given that PacBio has been used extensively for de novo assembly, this is unexcusable. Furthermore, the paper fails to mention that PacBio is very different in its read characteristics, particularly read length.A section on the experimental workflows for these systems attempts to summarize all of them in one paragraph. There is one serious error here; in library preparation PCR is performed after ligation of adaptors and not before. More seriously, the described workflow does not apply to either of the single molecule systems which they have mentioned; neither uses PCR and Helicos didn't even have a ligation step.A brief summary of de novo sequence assembly algorithms has a few small errors (for example, while many implementation of overlap-layout-consensus (OLC) use k-mers to speed execution, k-mer analysis is not an inherent facet of the algorithm as the authors suggest). More serious is that only de Bruijn graph and OLC are discussed; string graphs are omitted and would be very relevant to the purported scope of this paper.For the paper, the authors downloaded a single dataset from the Assemblathon dataset, for Zebrafish (oddly described as \"fish species M.zebrafish\"). No explanation is given why this dataset was chosen. Some assembly runs involved removing low quality data from the dataset, but no explanation is given as to what criteria were used to define low quality or tools used to remove them. This relates to another gaping hole in the manuscript: numerous approaches for preprocessing data have been described in the literature, including read filtering, read trimming, error correction, paired end merging and k-mer based normalization; none of these topics are broached. This will become clearly unfortunate later in their manuscript.While the title promises a significant comparison of methods, the manuscript describes using only two programs: Velvet standing in for single compute node de Bruijn graph algorithms and Contrail for distributed computing de Bruijn graph assemblers. While a few other DBG assemblers are mentioned, the existence of other DBG assemblers which can run across multiple compute nodes are not (e.g. Ray, ABySS). Since the manuscript focuses on the Hadoop aspect of Contrail (which is the framework it uses to distribute the computing across multiple nodes), the paper could leave the unfortunate impression that this is the only attempt in the field, rather than one of many mechanisms (e.g. MPI)The authors begin by trying a sampling of k-mer values for both Velvet and Contrail using a 2X dataset. The method used for downsampling the dataset is not given (while the text promises that the Perl programs used are available on request, this should be seen as an unacceptably inadequate mechanism; at a minimum they must be supplementary materials but better would be deposition in a public code repository). They measure two figures-of-merit (N50 and maximum contig size), but plot only one of them (though this plot is the best single element in the paper). A justification for using a 2X sample, rather than a larger one, is not given. This opens the question whether a larger k-mer length might have worked better on a larger dataset.The authors proceed to try both programs on the entire dataset; Contrail succeeds but Velvet fails. Velvet fails again on 50% of the data if in paired end mode (though again, the method of downsampling is not given) but runs on that dataset in unpaired read mode. Velvet is tried also, in both modes, on a 25% dataset and succeeds. The authors present the figures-of-merit as a table, with no apparent order. Since Contrail (at least the version used) had only an unpaired mode, it is run only once. This data would be far more useful plotted as a graph as well, with the table sorted in some order relevant to the user, such as the 2X, 25%, 50% and 100% of dataset.A serious issue at this point is the author's choice of N50 and maximum contig length as their sole figures-of-merit, which they mistakenly label as measures of assembly quality. At no point do the authors attempt to assess the correctness of their assemblies, despite this being a standard method in assembler comparisons (such as the Assemblathon from which the authors obtained the data used). Both N50 and maximum contig length can be inflated by overly aggressive assembly that yields misassembly artifacts, and N50 can be inflated by the choice of a minimum contig length cutoff. Indeed, the authors fail to report a genome size their assemblies, and so these assemblies could represent only a fraction of the target genome.The authors observe that the 25% and 50% datasets gave similar results for their figures-of-merit, and observe that less data can give equal or better results. They appear to have not asked if this has been observed before (it has). Nor do they run Contrail on the subsampled datasets to see if the trend holds there as well.The issue of Velvet crashing on the larger datasets is presented as highly significant; indeed the conclusion is drawn that multi-machine programs such as Contrail are required for this data. This is highly unfortunate on two grounds.First, as noted before, the authors performed nearly no preprocessing of the data (other than the ill-documented poor quality read removal). Sequencing errors will enlarge the de Bruijn graph, so error correction or read trimming can reduce the memory requirements of an assembler. Paired end merging can similarly reduce memory requirements, albeit at some risk of telescoping small repeats. Merging is particularly relevant for Contrail, since it does not explicitly handle paired ends. K-mer based read normalization can greatly reduce memory requirements for assembly.Second, a number of programs have demonstrated assembly of vertebrate-scale short read datasets on single machines, indeed single machines with far less memory than the 1Tbyte found compute node used for Velvet in the paper. Examples include Minia, with a de Bruijn graph structure designed to be extremely memory efficient, and Readjoiner, which uses a string graph paradigm (which, as noted above, is a strategy ignored by the paper in the introduction).Finally, the authors fail to make any attempt to place this in a relevant modern context. Given that short reads from short inserts alone are mathematically incapable of assembling anything but the simplest plasmid or viral genomes, the current thrust in de novo assembly is assembling either entirely from long reads or integrating short reads with long reads or mate pairs to accurately yield long (increasingly, chromosome-scale) scaffolds. Failing to place the very limited findings of this manuscript in such a context could be characterized as a final failing.", "responses": [] }, { "id": "7606", "date": "27 Feb 2015", "name": "Surya Saha", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDe novo assembly of large eukaryotic genomes remains a challenging task even with the increasing availability of high quality long reads and short reads from paired-end and mate-pair libraries. An in-depth comparison of the performance of assemblers using multiple data sets and operating on widely used high memory multi-core systems versus distributed platforms can be a valuable contribution to the field. However, the authors fail to deliver on the promise of the paper on several counts. They present a limited and unsound comparison of only two assemblers (Velvet and Contrail) using two poorly selected datasets from a single species. The experiment design and analysis is replete with numerous errors, large and small. The Background section is superfluous and contains uncited historical details that are not applicable to the context of the paper. The authors chose to highlight sequencing technology (Figure 1) that is outdated by at least two years (Illumina Genome Analyzer) and not available any more (Helicos, Roche 454) while neglecting to mention the workhorses of sequencing cores today. There was no discussion of the widely used platforms like Illumina Hiseq or desktop sequencers like Illumina Miseq or Ion Torrent PGM. There are also many factual errors such as the maximum length of 50bp for sequences generated using Maxam-Gilbert method and costs/ read lengths of different sequencing platforms. Maxam-Gilbert sequences range from 250bp to 500bp or longer 1. The sequencing costs and read lengths are outdated for Illumina Genome Analyzer and incorrect for SOLiD. The authors describe the concepts of overlap-layout-consensus (OLC) and de Bruijn graph assembly but neglect to mention string graph 2 theory.The methods used for evaluation are reasonably well documented. But a majority of the description could have been moved to supplementary data leaving room for a more qualitative discussion of the motivation for the methods used in the paper. The authors chose to evaluate only two assemblers without giving an explanation of why these two were selected. Velvet was selected as an example of serial de Bruijn graph assembler and Contrail as a compute-distributed assembler. The dataset selected by the authors is from a Lake Malawi cichlid (Maylandia zebra or Metriaclima zebra) incorrectly named as M. zebrafish in the paper. The reason for selecting this particular dataset and not a range of genomes with varying complexity is not explained despite the impact on the evaluation. The two cichlid data sets have either very low coverage or very high coverage, both of which are detrimental for de novo assembly. The authors refer to Perl scripts that are not included in the paper. This is not acceptable given the availability and ease of use of code sharing platforms like Git. Such scripts can also be included in supplementary data as text files. The only summary statistics used for evaluating assemblies are N50 and maximum contig size. These are not informative with regards to the quality of the assembly and will have ramifications for analysis. Error correction is mentioned but no further explanation is given. None of the many other well-known preprocessing practices such as k-mer based normalization and merging of paired-end reads were used before de novo assembly. The authors attempt to assemble the full dataset as well as various sub-sampled versions using the two assemblers. The sub-sampling procedure is not described in the methods. The analysis in the Discussion section, like previous sections, has several shortcomings. The authors refer to the Assemblathon2 3 comparison of assembly algorithms which set the standard for metrics to use for evaluation of assemblers. The evaluation of assemblies from Velvet and Contrail is quite inadequate as they authors did not check the assemblies for errors. They did not validate the assemblies by checking for the presence of core genes as described in Assemblathon2 3 or by comparing to the published genome. The assemblies were simply evaluated for contiguity using N50 and maximum contig size, both of which can be improved with parameters that can potentially increase the number of misassemblies. The authors conclude that assemblers based on serial or non-distributed algorithms cannot be used for large scale denovo assembly due to out of memory errors in Velvet. Velvetg fails to complete due to lack of memory but they do not explore the issue further. Large de Bruijn graphs are often caused by presence of sequencing errors in the reads. Velvet may be able to assemble the given dataset once poor quality reads are filtered out. Merging of paired-end reads and k-mer-based normalization are also effective strategies to reduce memory requirements. It is also not clear why the authors did not perform assemblies with 50% and 25% unpaired dataset with Contrail. They would provide additional data points for comparison with Velvet even for the underpowered experimental design used in this paper. The information presented in this paper is outdated and the experiments and analysis are woefully inadequate to judge the effectiveness of serial versus distributed genome assemblers. Moreover, the paper does not utilize or even address the commonly used approach of combining long reads with higher coverage paired-end and mate-pair short reads to generate assemblies for large eukaryotic genomes.", "responses": [] } ]	1	https://f1000research.com/articles/4-20
https://f1000research.com/articles/4-19/v1	22 Jan 15	{ "type": "Opinion Article", "title": "Every scientist is a memory researcher: Suggestions for making research more memorable", "authors": [ "Christopher R. Madan" ], "abstract": "Independent of the actual results, some scientific articles are more memorable than others. As anyone who has written an article collaboratively knows, there are numerous ways a manuscript can be written to convey the same general ideas. To aid with this, many scientific writing books and editorials provide advice, often anecdotal, on how to make articles more memorable. Here I ground these suggestions with empirical support from memory research. Specifically, I suggest that researchers consider how to emphasize their work’s novelty, strive to describe their work using concrete, easy-to-understand terms, and use caution when attempting to evoke an emotional response in the reader. I also discuss considerations in title selections and conference presentations.", "keywords": [ "scientific writing", "memory", "citation frequency", "scholarly communication", "publishing" ], "content": "Introduction\n\nEvery scientist wants their paper to be read by their peers, their theories and results to be remembered, and their work to be cited in subsequent papers. Many factors influence the likelihood that a paper will be read and later cited by a reader, such as the relevance of the topic to the reader’s own interests, the journal that the work was published in, the quality of the experimental design (e.g., sample size, statistical rigor), and the work’s exposure in the media and social media (i.e., altmetrics) (Bartneck & Hu, 2009; Callaham et al., 2002; Eyre-Walker & Stoletzki, 2013; Fraley & Vazire, 2014; Leimu & Koricheva, 2005; Piwowar, 2013). (While the impact factor of the journal an article is published in influences the likelihood of the work being read by other researchers, the citation frequency of the work itself is definitively a more relevant measure of an article’s impact, e.g., Brembs et al., 2013; Editorial, 2003; also see “Measuring the Impact of Scientific Articles” by Peterson). In addition to these factors that are more generally acknowledged as influencing citation frequency, other factors also correlate with citation frequency, such as gender, number of co-authors (particularly those from other institutions), and citation networks (i.e., research groups that often cite each other) (Borsuk et al., 2009; Campbell et al., 2013; Maliniak et al., 2013). In addition to these factors, other factors can further influence citation frequency, such as memorability. Though memorability may co-vary with some of the other factors mentioned, specific strategies, grounded in the human memory literature can be used to make a research paper more memorable.\n\nA paper’s memorability is more difficult to quantify than many other factors; however, it could be viewed as even more important since the researcher may have more control over it. A researcher should not change their research topic to a ‘hotter’ topic simply for the sake of garnering more citations, nor should they seek out co-authors from other institutions solely in the hopes of garnering more citations. However, there is often more than one way to write a paper on a given research study, depending on how the topic is related to the relevant background literatures. It is also possible to make a paper more readable by using more direct language when describing the experimental design and results (see Dunleavy, 2003; Pinker, 2014; and Kail, 2014, for detailed discussions of scientific writing). However, the focus of the current paper is on grounding anecdotal scientific writing advice in the human memory literature, providing empirical support for making a paper more memorable. (I should state that my papers likely have not capitalized on these memory principles as well as they should have—connecting scientific writing to memory findings has been a learning experience for me as well). Many of these scientific writing suggestions and related memory findings are listed in Table 1.\n\n\nBe novel\n\nOne of the most robust and well-known findings in the memory literature is the serial position curve (Ebbinghaus, 1885; Hasher, 1973; Murdock, 1962). Briefly, if a list of items is presented to participants and they are subsequently asked to recall all of the items from the list that they can, the participants are more likely to recall the first and last items in the list, relative to the intermediate items. These two effects are respectively referred to as the primacy and recency effects. With respect to citation frequency, Newman (2009) has reported a “first-mover advantage”, suggesting that a researcher publishing a modest paper on next year’s hot topic will accrue more citations than if they had instead published an outstanding paper on the current year’s topic. This difference in citation frequency could be attributed to a primacy effect, where other researchers are more likely to remember the first paper on a topic because it was the first and is thus more easily sampled from memory when thinking of seminal studies of a topic. Furthermore, the first paper published on a topic is more novel than its contemporaries. A large body of memory research supports this notion of memory enhancement for novel information (Gabrieli et al., 1997; Wittman et al., 2007).\n\nAnother way of viewing effects of novelty on memorability is that novel information is more distinctive. While other studies may demonstrate modest advances in our understanding of a topic, novel studies stand out and take the field forward more significantly. The finding that distinctive information is remembered better is known as the isolation effect or the von Restorff effect (von Restorff, 1933). In an experimental setting, the von Restorff effect is often studied by presenting participants with a list of words sequentially, with one word being more distinct in a perceptual (e.g., font color, font size) or conceptual (e.g., emotional, semantic category) dimension. In terms of scientific writing, a researcher likely reads many papers over the course of several months, and one that is distinctive and stands out from the rest is more likely to be subsequently remembered and later cited when the researcher is writing their own manuscript.\n\nWhile memory findings can help explain why novel research is likely to be remembered and cited, making a study appear novel is not as straightforward. Some researchers are quite adept at discovering new advances in their fields, such as by integrating ideas across disciplines, and the resulting products are often novel. Indeed, analyses of high-impact papers have found that multidisciplinary teams are more likely to produce novel and innovative advances (Uzzi et al., 2013). For some guidance on coming up with novel research ideas, see Yewdell (2008).\n\n\nBe interesting\n\nAnother common suggestion for scientific writing is to make the paper ‘interesting’ (e.g., Bartunek et al., 2006; Davis, 1971; Gray & Wegner, 2013; Sand-Jensen, 2007). Gray & Wegner (2013) suggest six guidelines for making one’s research interesting. While summarizing their eloquently described guidelines does them a disservice, briefly, they make three suggestions related to choosing the research question and three related to the how the researcher goes about answering the question. They suggest that a researcher choose a research question that (1) focuses on the phenomena of interest first, rather than extant studies and theories; (2) is surprising, study phenomena that are counter-intuitive (“If results were exactly as predicted, would they be interesting”?); and (3) would interest a layperson and not just researchers (but note: “Conversely, countering laypeople’s intuitions may yield fewer immediate citations, especially if the research does not easily fit into established scientific paradigms”). On the other side, they suggest that the researcher addresses the research question using an experiment that (4) would be engaging to the participant; and (5) lends itself to simple statistics (“If a more complicated analysis is needed, think about redesigning the study; 4-way interactions can be explained, but would anyone care enough to listen?”). Finally, Gray and Wegner suggest that (6) the paper be written in a way that emphases it’s importance and the universal truths that it is built upon are clear and evident to a layperson, not just researchers. (Also see Bartunek et al., 2006, for an empirical analysis that converged on similar reasons of why an article was found to be interesting by readers).\n\nGray and Wegner’s guidelines can map on to several memory-relevant mechanisms. The idea of surprise corresponds well with the suggestion of making the work appear novel and distinctive from prior published studies. They also suggest that writing be generally more understandable and engaging. One implication of this suggestion is to describe the motivation and interpretation of the study using more concrete and imageable terms, rather than abstract concepts. In this regard, memory studies have definitive evidence that concrete words are remembered better than abstract words (Paivio, 1971; Paivio, 1986). Evidence also shows that imageability enhances association learning (Madan et al., 2010), which may make it easier to remember the links between different ideas within the paper later on. Providing illustrations of concepts (e.g., hypothesized results; theoretical models) may also help in this regard.\n\nThe notion that writing compelling and engaging prose would increase memorability could also be thought of as adding an emotional arousal component. Emotional content itself is often better remembered (Kensinger & Corkin, 2003; Talmi & Moscovitch, 2004). However, in contrast to imageability, which enhances both item- and association-memory, the effects of emotional arousal on memory appear to be more of a double-edged sword. Specifically, while emotional items (e.g., words, images) tend to be better remembered than neutral items, this often is at the cost of memory for the context that the emotional items are learned in. If the context is intrinsic to the emotional item (e.g., font color), memory for the associated information may be enhanced, however, when the context is more separable, such as two unrelated words, memory for the association is impaired (Kensinger, 2009; Madan et al., 2012; Mather & Sutherland, 2011). With respect to scientific writing, if an amusing anecdote is used to help motivate the research question, the anecdote—as an emotional item—may be remembered well, but if too unrelated to the actual topic of the paper, the reader may later have trouble recalling where they read the anecdote.\n\nAdditionally, Gray and Wegner rightly comment that “People have a powerful memory for endings (Kahneman et al., 1993), and so you want the reader to remember your paper with a tinge of giddiness and awe”. Indeed, Kahneman et al. (1993) found that in patients undergoing a colonoscopy, post-operative pain judgments were correlated with the peak intensity of pain (as judged in real time) and with the pain intensity at the end, but not with the duration of the procedure. Delayed judgments relating to positive experiences, such as vacations, are similarly influenced by peak and end intensities (Mitchell et al., 1997). This phenomenon, known as the peak-ends rule, demonstrates that people’s memories of an experience are biased, with the ending being particularly important.\n\n\nComments on selecting a title\n\nWhen evaluating an article’s relevance, the reader likely first starts with the title. Studies of citation frequencies have yielded mixed results, with some finding higher citation rates associated with longer titles (Habibzadeh & Yadollahie, 2010; Jacques & Sebire, 2010), others suggesting the use of shorter titles (Paiva et al., 2012; Subotic & Mukherjee, 2014), and others yet observed no relationship between title length and citation rates (Jamali & Nikzad, 2011). Memory research suggests that longer words are harder to remember (Baddeley et al., 1975), but the title of a research paper is more complex—the string of words that comprise a title have meaning. Along these lines, papers with titles that described the study’s results had higher citation rates (Jamali & Nikzad, 2011; Paiva et al., 2012). As examples of this, “Articles with short titles describing the results are cited more often” (Paiva et al., 2012) and “Emotional arousal does not enhance association-memory” (Madan et al., 2012). However, there is evidence that questions directly describing the research question may have higher citation rates (Jamali & Nikzad, 2011), e.g., “Is the enhancement of memory due to reward driven by value or salience”? (Madan & Spetch, 2012). Question-based titles are more likely to engage the reader to think about the research question for themselves, relative to titles that more generally describe the research topic or state the main result. This is particularly important since people remember more about information they think about elaboratively, as found in the levels-of-processing framework (Craik & Lockhart, 1972).\n\nFocusing more on the content, many suggest that a title be ‘catchy’ (Atkin, 2002; DeBakey, 1977; Schultz, 2009). While this is a good suggestion at face value, one must subsequently consider—what makes a title catchy? Atkin (2002) focused on phrases that suggested innovation, specifically “paradigm shift” and “pushing the envelope”. While these phrases may be catchy, the also should not be over used. Schultz (2009, p. 21) describes the purpose of the title as: “The title is your first opportunity to attract an audience to your paper. A well-worded and catchy title can lure reluctant readers to take a closer look at your paper”. More completely, Schultz suggests that titles should be informative, accurate, clear, concise, and attention commanding (see Schultz, 2009, for further details).\n\nRegardless of one’s opinion of catchy titles, it is important to consider if the potential title is informative (Comroe, 1966; DeBakey, 1977; Dunleavy, 2003; Eva, 2013; Hartley, 2005; Kazdin, 2013; Mermin, 2003; Paiva et al., 2012; Schultz, 2009; also see “Why do academics and PhDers carefully choose useless titles for articles and chapters?” by Dunleavy). While informative titles tend to be longer, they also are more likely to attract readers who are interested in the topic. As an example, Hartley provides a catchy title that was assigned to an article of his (“More sex please, we’re psychologists”) along with his original title (“Were there any sex differences? Missing data in psychology journals”). Clearly, these titles are not equally informative of the topic of the article, providing an example of when aiming for a catchy title can go too far. If a title is catchy but not informative, it is unlikely to attract readers who are indeed interested in the topic (DeBakey, 1977). Ideally, catchy titles can still be informative. For instance, “Short and amusing: The relationship between title characteristics, downloads, and citations in psychology articles”. (Subotic & Mukherjee, 2014) and “Building a memory palace in minutes: Equivalent memory performance using virtual versus conventional environments with the Method of Loci” (Legge et al., 2012). In many fields, the use of colons in titles has been found to be fairly common, with papers not using colons being cited less often; however, this effect varied greatly between disciplines and was even reversed in some (Buter & van Raan, 2011).\n\nSeveral other title properties have also been considered. For instance, a title’s perceived amusement has been found to either have no influence on citation rates (Subotic & Mukherjee, 2014) or possibly a negative effect (Sagi & Yechiam, 2008). Pleasantness is positively correlated with citation frequency (Sagi & Yechiam, 2008), though it is not clear if emotion-memory effects mediate this relationship. Whissell et al. (2013) measured trends in titles over the last fifty years and observed shifts towards using more concrete and emotional words in more recent years.\n\n\nPresentation-specific advice\n\nWhile the notions of communicating research such that it is novel and interesting are important regardless of whether a project is written as a manuscript or presented at a conference, some advice is more specific to presenting research. Written firmly tongue-in-cheek, (Wolcott, 1997a; Wolcott, 1997b) provides many great suggestions for poster and oral presentations. (The original versions of these newsletter articles have been archived online: poster [1997a], oral [1997b]). Briefly, in an aptly titled article, “Mortal sins in poster presentations or, How to give the poster no one remembers”, Wolcott (1997a) suggests poster presenters consider how to logically layout and color the poster, and select an informative title. Most importantly, Wolcott (1997a) provides advice on formatting text to enhance readability, e.g., size, spacing, concise phrasing, organization, and color scheme. See Block (1996) for more poster-related advice. For selecting colors that are distinguishable by color-blind readers, see Wong (2011).\n\nWhen discussing oral presentations, Wolcott (1997b) gives some similar advice, such as that the content on the slides be easy to read, not overwhelming, and focus on the bigger picture ideas rather than staying too close to the results. Additionally, presenters should make sure to talk to their audience (as opposed to talking at the projector screen), practice the presentation with a friendly audience, be aware of the contents of upcoming slides, and speak sufficiently loudly. See Harolds (2012) and Schultz (2009, especially p. 284) for additional advice for oral presentations.\n\nAs evident by Wolcott’s suggestions, presenting one’s research involves additional facets that are present in manuscripts. For instance, text in manuscripts must formatted following a journal’s guidelines, preventing font selection, color, and spacing from being potential issues. Furthermore, manuscripts must follow standard organizational structures (e.g., Introduction, Methods, Results, Discussion). In contrast, both poster and oral presentations involve these additional decisions, which may result in possible missteps. With respect to making your presentation memorable, if the audience is unable to follow along with the presentation, e.g., the presenter is not sufficiently audible or if the slides/poster content distracts rather than serves as a visual aid, then the presentation will be less memorable. Furthermore, other factors that may simply not have crossed the mind of the presenter may also have a significant effect on in-person presentations, but be irrelevant to manuscripts. For instance, Keegan & Bannister (2003) found that when a presenter wore attire that was color coordinated with their poster, more visitors came to the poster.\n\nThe flexibility that presentations afford also provides additional opportunities to make research memorable. For instance, while concrete words are remembered better than abstract ones, memory for pictures is superior (Paivio & Csapo, 1969; Paivio & Csapo, 1973). Additionally, images that visualize information can convey more information, improving comprehension of the ideas they represent (Robinson & Kierwa, 1995). Images can also be used more flexibly in presentations; for instance, images that are bizarre or humorous are remembered particularly well, likely due to a distinctiveness-based mechanism (McDaniel & Einstein, 1986).\n\nKosslyn et al. (2012) provides additional presentation-related advice, grounded in psychological theory. Kosslyn has also published two books on this topic, providing a wealth of suggestions (Kosslyn, 2007; Kosslyn, 2011).\n\n\nFurther reading\n\nI hope that grounding scientific writing advice in memory literature was useful in demonstrating the mechanisms whereby a research paper could be made more memorable. Nonetheless, I do not think this is a sufficient substitute for concise and well-written advice. In particular, I strongly suggest the reading of: Gray & Wegner (2013); Sand-Jensen (2007). Hengl and Gould’s brief guide to writing research entitled “Rules of thumb for writing research articles” is also a must-read.\n\nFor readers interested in additional advice on scientific writing and presenting: Kazdin (2013) provides a particularly well-written and concise summary of the necessary details to include when preparing a manuscript, as well as advice on selecting a journal and navigating the peer-review process. Dunleavy (2003) provides a comprehensive discussion of the scientific writing process and its related considerations. For further advice for poster presentations, see Block (1996) and “Creating Effective Slides Without Having to Become a Graphic Designer”. For more comprehensive guides on scientific presentations and speaking about science in public, see Albuquerque (2015); Alley (2013); and Meredith (2010).", "appendix": "Competing interests\n\n\n\nThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\n\n\nGrant information\n\nCRM was supported by a National Science and Engineering Research Council (NSERC) Alexander Graham Bell Canada Graduate Scholarship (Doctoral level).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThis work was inspired by workshops by Robert Kail and Jay Van Bavel at the University of Alberta in the summer of 2013.\n\n\nReferences\n\nAlbuquerque UP: Speaking in Public About Science. New York: Springer. 2015. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "7411", "date": "09 Feb 2015", "name": "Daniela Palombo", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDr. Madan has written an insightful and useful paper that can benefit writers in many fields of science. Grounding the paper's theme in memory phenomena is clever. I suggest the article be approved with reservations.Here are my specific recommendations: Dr. Madan describes the \"first mover advantage\" (Newman, 2009): \"a researcher publishing a modest paper on next year’s hot topic will accrue more citations than if they had instead published an outstanding paper on the current year’s topic.\" Dr. Madan suggests this may have to do with a primacy effect in memory. While this may be true, another obvious explanation for the \"first mover advantage\" is that the researcher simply misses the boat. Coming out before the wave means all those topical papers that will be published during the wave will cite you ---simply because it's a new topic and your paper is published and there may be little in the area. If you miss the wave, you miss out on those citations. So this is less attributable to memory but more about timing. This is another explanation that might be worth noting. Under \"be interesting\" I would stress the importance of transparency in writing a bit more. In my opinion, better remembered articles are those that I understood enough to be able to mention to other colleagues and explain to them fully. If an article has either too much technical jargon, is too brief, or is too complicated (as you note), it won't be fully comprehended and unlikely to be retained (and cited--no one wants to cite a paper they don't understand for fear you are not citing it accurately). Dr. Madan touches on these issues but I feel they can be emphasized more, as there is sometimes a tradeoff between sounding interesting and being clear. Both are important but one should not sacrifice the other.I recommend after this sentence: \"This phenomenon, known as the peak-ends rule, demonstrates that people’s memories of an experience are biased, with the ending being particularly important\", that the author includes something a bit more concrete as a recommendation for capitalizing on recency. Perhaps it would be helpful for the author of a manuscript to summarize really clearly the main findings and implications of the study with a very powerful take-home message because that will be the last thing the reader remembers. I feel this is true for some of the other instances were the author draws on the memory literature in that more examples relevant to manuscript writing process would be helpful. The section on presentation specific advice is not quite as well developed as the section on manuscript writing. That is ok, in principle, since the paper's main focus is on written work. However, I do find I take less away from that part of the paper. I may consider beefing it up a bit. One recommendation I have regarding poster presentations is that they should be prepared with two types of visitors in mind: Visitor A is someone who sought the poster out and wants the full explanation from the presenter and will follow along on the poster as the presenter is speaking. Visitor B is someone who is moderately interested in the poster and wants to be able to get the \"gist\" from it in only a few minutes. The poster should stand on its own so that if Visitor B just wants a reprint they would be able to figure out what the study was about with fairly little effort. Otherwise, the paper sounds great.", "responses": [] }, { "id": "7789", "date": "24 Feb 2015", "name": "Alan Castel", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting and novel paper, with some very informative data! I have two general comments 1) sometimes scientists are not good at knowing what is interesting/clear due to the curse of knowledge, and that could be tied into this paper (perhaps outsiders are better at choosing interesting titles or summarizing research findings?) 2) there is a strong metacognitive component regarding knowing what other people may or may not remember, and there is a lot of work showing that sometimes rememberers are not good judges of what is memorable. The author talks about primary and recency effects but sometimes people even fail to incorporate serial position effects when making memorability judgments, albeit when studying lists of words (e.g., Castel, 2008), or fail to consider the effects that long retention interval have on memory (see much of Asher Koriat’s work), even when we might be explicitly aware of these issues in other contexts. Suffice to say that people aren’t always the best judges of what is memorable, and this might also extend to titles of articles or how to make article memorable. I think these two points could be considered, but overall I enjoyed reading this paper (and won't forget it)!", "responses": [] }, { "id": "7788", "date": "09 Mar 2015", "name": "Mathieu Hainselin", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper is interesting for researcher, and I recommend its indexing with some possible modifications. I think, as a memory researcher, that this paper follows most of its own advice, and explains in a simple and concrete way how to improve manuscripts.Although using all those advice is not easy, as the author writes himself, I think this paper would improve with some concreteness. For example, an example could be added after “With respect to scientific writing, if an amusing anecdote is used to help motivate the research question, the anecdote—as an emotional item—may be remembered well, but if too unrelated to the actual topic of the paper, the reader may later have trouble recalling where they read the anecdote.” or “Evidence also shows that imageability enhances association learning (Madan et al., 2010), which may make it easier to remember the links between different ideas within the paper later on. Providing illustrations of concepts (e.g., hypothesized results; theoretical models) may also help in this regard.” Likewise, less likely to be retained titles for this paper could help those who aren't (yet) memory researchers to see the differences between “good” and “bad” titles.The sentence “In terms of scientific writing, a researcher likely reads many papers over the course of several months, and one that is distinctive and stands out from the rest is more likely to be subsequently remembered and later cited when the researcher is writing their own manuscript.” might be speculative. If there is no doubt concerning distinctiveness, the correlation between this memory effect and citations would need more references or some discussion.More explicit explanations and examples concerning the repetition effect could also be interesting for the readers. Should authors write the same (main) message in the article?In the end, despite minors remarks, the paper is very interesting and will probably be useful for many researchers.", "responses": [] } ]	1	https://f1000research.com/articles/4-19
https://f1000research.com/articles/4-18/v1	22 Jan 15	{ "type": "Software Tool Article", "title": "FORGE: A tool to discover cell specific enrichments of GWAS associated SNPs in regulatory regions", "authors": [ "Ian Dunham", "Eugene Kulesha", "Valentina Iotchkova", "Sandro Morganella", "Ewan Birney", "Eugene Kulesha", "Valentina Iotchkova", "Sandro Morganella", "Ewan Birney" ], "abstract": "Genome Wide Association Studies (GWAS) provide an unbiased discovery mechanism for numerous human diseases. However, a frustration in the analysis of GWAS is that the majority of variants discovered do not directly alter protein-coding genes. We have developed a simple analysis approach that detects the tissue-specific regulatory component of a set of GWAS SNPs by identifying enrichment of overlap with DNase I hotspots from diverse tissue samples. Functional element Overlap analysis of the Results of GWAS Experiments (FORGE) is available as a web tool and as standalone software and provides tabular and graphical summaries of the enrichments. Conducting FORGE analysis on SNP sets for 260 phenotypes available from the GWAS catalogue reveals numerous overlap enrichments with tissue-specific components reflecting the known aetiology of the phenotypes as well as revealing other unforeseen tissue involvements that may lead to mechanistic insights for disease.", "keywords": [ "Genome wide association", "GWAS", "Hypersensitive sites", "regulatory elements" ], "content": "Introduction\n\nA primary motivation for sequencing the human genome was to shed light on mechanisms involved in human disease. Since finishing the sequence there has been much activity in two areas towards that goal. In the first, extensive re-sequencing of individual genomes has provided comprehensive lists of human variations, which can in turn be examined for association with disease and other phenotypes in Genome Wide Association Studies (GWAS)1. In the second area, efforts have been undertaken to identify the specific sequences that enact function within the genome including, but not restricted to, regions defining genes and their controlling elements2–4. The aim, of course, is to understand the associations uncovered in the first approach in the context of the annotations delivered from the second.\n\nThe past few years have seen a dramatic growth in the number of variants associated with disease by GWAS1. An extensive catalogue of GWAS associations has been compiled containing nearly 14,000 associations of variants to phenotypes5. However, a crucial observation is that the majority of the variants observed do not directly affect the coding regions of protein coding genes. Notwithstanding that the reported variant for an association may be in linkage disequilibrium with a causal variant affecting a protein coding sequence, regulatory regions have been demonstrated to be linked to both specific diseases associations6–18 (see 19 for review and further examples) and to be enriched in bulk in SNPs found across all GWAS2,20–22. The ENCODE consortium reported that GWAS single nucleotide variants are substantially enriched in regulatory regions and up to 80% of GWAS variants have a potential regulatory interpretation via overlap with regulatory annotation2,21,22. Furthermore, Maurano et al.21 showed that regulatory regions revealed by the DNase-seq method show a cell-specific enrichment for GWAS variants in specific phenotypes consistent with probable physiological mechanisms. Trynka et al.23 similarly found that regulatory elements identified by the histone modification H3K4me3 show a phenotypically relevant cell-specific overlap with GWAS SNPs. Several tools exist to highlight the specific overlaps of individual GWAS SNPs with potential regulatory regions24,25. To date however, much of the focus on this work has been on prioritising variants, rather than exploring the extensive cell type information present in the large-scale projects.\n\nWe have developed a simple but powerful approach that identifies significant cell-specific enrichments in regulatory regions for sets of single nucleotide variants, typically from GWAS. We name the approach Functional element Overlap analysis of the Results of GWAS Experiments or FORGE, and have implemented it as both a rapid web tool for ENCODE26 and Roadmap Epigenome project DNase-seq data27 and a free-standing open source software. The web tool produces two alternative graphical outputs for exploration alongside tabulated enrichment data. FORGE analysis across all eligible phenotypes in the entire GWAS catalog5 identifies numerous interesting patterns of enrichment by cell type and suggests tissues to focus on for future follow up studies.\n\n\nMethods\n\nENCODE consortium hotspots26 were obtained from ftp://ftp.ebi.ac.uk/pub/databases/ensembl/encode/integration_data_jan2011/byDataType/openchrom/jan2011/combined_hotspots/. Roadmap Epigenome DNase1 sequencing tag alignments were obtained from http://www.genboree.org/EdaccData/Current-Release/experiment-sample/Chromatin_Accessibility/. The files used correspond to that part of the Gene Expression Omnibus (GEO) accession GSE18927 beyond the data use embargo date. These alignments were processed by the Hotspot (http://www.uwencode.org/proj/hotspot/)28,29 method with the default parameters to give hotspot and peak files. For this analysis we choose to use the hotspots which are regions of general DNase I sensitivity rather than peaks which are more similar to DNase I hypersensitive sites because, although the method works with peaks, hotspots reveal more tissue specific signal (data not shown). Cell and tissue assignments for each of the datasets were made using the decodings available from the ENCODE Data Coordination Center tables (https://genome.ucsc.edu/encode/cellTypes.html) or from the BioSamples database (http://www.ebi.ac.uk/biosamples/) sampleGroup SAMEG31306. A list of samples used is provided in Supplementary Table S1.\n\nThe complete collection of SNPs discovered in GWAS studies curated in the NHGRI GWAS Catalog5 were downloaded from http://www.genome.gov/gwastudies/30 (Accessed 3rd September 2014). SNPs were grouped according to the annotation provided in the Disease/Trait field and only sets with 5 or more non-redundant SNPs were retained. See Supplementary Table S2 for list of SNP sets analysed. A set of files of the SNPs included in analysis for each phenotype is available in the data directory of the GitHub release, https://github.com/iandunham/Forge. For FORGE analysis SNP sets were further filtered by LD pruning removing all but one SNP from a set of SNPs at r2 >= 0.8 in the 1000 genomes data (see below) and were analysed for both ENCODE and Roadmap Epigenome DNase I hotspots, selecting background SNP sets from the default GWAS genotyping array SNPs.\n\nThe FORGE tool utilises either an SQLite (command line tool, http://www.sqlite.org) or MySQL (web tool, http://www.mysql.com) database of the overlaps of every 1000 genomes project (http://www.1000genomes.org)32 SNP with the ENCODE and Epigenome Roadmap DNase1 hotspots. To prepare this database, SNPs from the 1000 genomes phase 1 integrated call data set were downloaded from ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/phase1/analysis_results/integrated_call_sets and compared to indexed DNase I hotspots using tabix from the SAMtools package (http://samtools.sourceforge.net/tabix.shtml)33 using a distributed approach on the EBI compute farm. The overlaps for each SNP were stored in a single large indexed table of SNP location and identifier with binary strings representing the presence (1) or absence (0) of overlap in each sample for each of the hotspot datasets (ENCODE or Roadmap).\n\nTo prepare sets of background SNPs matched to the test SNP set, FORGE matches SNPs based on G+C content (GC), minor allele frequency (maf) and transcription start site (TSS) distance, and repeats the overlap analysis for each of 1000 background sets. Overall population mafs were obtained from the 1000 genomes project phase 1 integrated call dataset at ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/phase1/analysis_results/integrated_call_sets. To control for the processes involved in selecting SNPs for genotyping, only 1000 genomes phase 1 SNPs that had been included on one of the common genotyping platforms as described in Ensembl (http://www.ensembl.org/info/genome/variation/data_description.html#variation_sets) were considered further. This left either 1875813 SNPs across various platforms (Affy GeneChip 100K Array, Affy GeneChip 500K Array Affy SNP6, HumanCNV370-Quadv3, HumanHap300v2, HumanHap550v3.0, Illumina Cardio Metabo, Illumina Human1M-duoV3, Illumina Human660W-quad) or 2231212 SNPs across the Illumina HumanOmni2.5 array. TSS distance was determined for each remaining SNP relative to the TSS defined by the Gencode project34,35 given in ftp.ebi.ac.uk/pub/databases/ensembl/encode/integration_data_jan2011/byDataType/gencode/jan2011/Gencodev7_CAGE_TSS_clusters_June2011.gff.gz using Bedops closest-features36. GC was determined for a 100 bp window centred on the SNP at base 50. The SNPs were then sorted into 1000 bins partitioned by deciles for each of GC, maf and TSS. For each SNP in a test set, the corresponding bin is identified based on its GC, maf and TSS distance, and background selections are made from that bin.\n\nA set of SNPs can be presented to FORGE as a list of RSIDs or by genome location on human genome build GRCh37 in either VCF or Bed formats. If RSIDs are not provided in one of these formats, the genome coordinates are used to identify the RSID. SNPs not present in the 1000 genomes phase 1 integrated call dataset are excluded from the analysis. With LD pruning selected a single SNP (the first in the file) is chosen from LD clusters within either r2 >= 0.8 or r2 >= 0.1 as specified. For each analyzable SNP in the test set, overlaps are retrieved from the FORGE database, and a count of total hotspot overlaps is recorded for each DNase I sample (cell) for the test SNP set. One thousand matching background SNP sets containing the same number of SNPs as the test SNP set are selected, matched for GC, maf and TSS distance by decile bins as described above. Overlaps for each of the SNPs in each of the background SNP sets are also retrieved from the database and an overlap count for each background set in each DNase I sample is recorded. For each test SNP set, the background probability of overlap is determined from the 1000 background set overlap counts and the probability of the observed test result under a binomial distribution is calculated. The P value thresholds of 0.05 and 0.01 are corrected for multiple testing by division by the number of tissue groupings tested, and the corrected threshold is used. The use of tissue as the unit for sample grouping is consistent with the groupings obtained by hierarchical clustering of samples by DNase I data (results not shown). The corrected thresholds are therefore more stringent than established by the random trials.\n\nFORGE generates both tabular and graphic descriptions of the enrichment of overlap for the test SNPs for each DNase I hotspot sample. A tab-separated values (TSV) file is output including columns for the binomial P value, cell, tissue, filename of the sample hotspots, SNPs that contribute to the enrichment, and the GEO accession for each sample. These data are also provided as an interactive table produced using the Datatables (https://datatables.net/) plug-in for the jQuery Javascript library accessed through the rCharts package (http://ramnathv.github.io/rCharts/).\n\nEach of the graphic outputs presents the –log10 binomial p by cell sample. A pdf graphic is generated using base R graphics (http://www.r-project.org‎). The interactive Javascript graphic is generated using the rCharts package (http://ramnathv.github.io/rCharts/) to interface with the dimple d3 libraries (http://dimplejs.org). In both cases cells are grouped alphabetically by tissue, and for the pdf alphabetically by cell. The interactive graphic stacks replicate samples at the same x coordinate. In each of the graphics the colouring of results by P value is consistent, blue (p > 0.05 equivalent after correction), pink (0.05 => p < 0.01), and red (p <= 0.01). The corrected P value threshold is given on the pdf output.\n\nTo estimate false positive rates, 1000 sets of SNPs at each of a series of SNP counts between 5 and 300 SNPs were randomly chosen from the 1000 genome phase 1 integrated SNP set. FORGE analysis was run for each set across the ENCODE and Roadmap Epigenome data, and the number of tests with P values less than thresholds ranging from 0.05 to 0.001 were recorded. These represent the false positives from 1000 trials at each of 424 samples i.e. 424,000 tests, and were used to calculate false positive rates at each significance threshold.\n\nThe hierarchical clustering solution was obtained using a multi-scale bootstrap resampling approach. We first computed a binary regulatory signature for each cell type classifying each DNase I site as active or inactive in each cell type sample. Hierarchically clustering of the binary regulatory signatures was by Euclidean distance with Ward's agglomerative method using the pvclust R/CRAN package with default values (http://cran.r-project.org/web/packages/pvclust/index.html). Finally, we identified clusters supported by the data at a bootstrap probability p value < 0.01.\n\n\nResults\n\nFORGE analysis provides a method to view the tissue-specific regulatory component of a set of variants. In its current implementation, FORGE analysis takes a set of single nucleotide polymorphisms (SNPs), such as those SNPs reported above the genome wide significance threshold (p < 5e-8) in a GWAS study, optionally filters the SNPs to remove all bar one SNP from a region in high LD (“LD pruning”) and determines whether there is enrichment for overlap with putative regulatory elements compared to a matched background of SNP sets. Initially the elements considered are DNase I hotspots generated from either the ENCODE26 or Roadmap Epigenomics projects DNase I data by the Hotspot method28,29, because of both the comprehensiveness of the sites identified and the broad range of cell types for which DNase I data were available. DNase I hotspots can be regarded as regions of general DNase I sensitivity.\n\nFor each set of test SNPs, an overlap analysis is performed against the DNase I hotspots for each available cell sample separately (125 samples for ENCODE, 299 for Roadmap, described in Supplementary Table S1), and the number of overlaps is counted. Major potential confounders in this analysis are the many biases of GWAS SNP distribution on the genome. To account for this a background distribution of the expected overlap counts for this SNP set is obtained by identifying 1000 matched background SNP sets of the same number of SNPs, matching each test SNP with an equivalent SNP by decile bin for each of G+C content (GC), minor allele frequency (maf) and distance to the nearest transcription start site (TSS). The matched background SNPs sets are overlapped with the DNase I hotspots and the background distribution of overlap counts is determined. The enrichment of the test SNP set for each sample is expressed as the binomial P value of the test SNP set given the background overlap distribution. The FORGE results are presented in interactive and static graphical and tabular forms by cell type. Enrichments above the background distribution with binomial P values less than 0.01 corrected for multiple testing are considered significant and are highlighted in red in the graphical output. Enrichments with p <= 0.05 are also highlighted in pink (Figure 1). As the DNase I patterns are not independent between cell types, we conducted simulation experiments with randomly selected input SNPs. We chose 1000 random test SNP sets for each of a series of SNP counts ranging between 5 and 100 SNPs and conducted FORGE analysis on both ENCODE and Roadmap data. The false positive rate was determined as the number of cell type enrichments identified greater than the significance thresholds used by FORGE expressed as the proportion of the total number of sample overlap tests performed (424,000) for each SNP count. This analysis showed that the P value thresholds are reasonably well calibrated to false positive levels around 0.5–0.75 %. In practice we correct the thresholds further for multiple testing across different tissues so as to be more stringent and so expect typical false positive levels to be less. As discussed below, many of the GWAS SNP sets do not reveal any enrichment, consistent with low false positive rates.\n\nA series of FORGE analysis results are presented for autoimmune phenotype GWAS SNPs. Each point represents the -log 10 binomial P value (y axis) of the enrichment of the test SNP set compared to matched background SNPs on a single DNase I hotspot sample, organized by tissue as indicated by the brown labels at the top of the figure, and alphabetically by cell sample (x axis). Where informative, additional labels at the bottom of the figure highlight relevant distinct cell types. Red points are at p <= 0.01 equivalent after multiple testing correction, pink points at 0.05 => p < 0.01. Full lists of the cells and results for each analysis are available in the Github data directory at https://github.com/iandunham/Forge. Phenotypes are labeled beneath each result.\n\nWe have implemented FORGE as a web tool available at http://browser.1000genomes.org/Homo_sapiens/UserData/Forge. The interface accepts a list of SNPs by dbSNP RefSNP identifier (RSID) or by genomic location on human genome build GRCh37 in either Variant Call Format (VCF, http://www.1000genomes.org/wiki/Analysis/Variant Call Format/vcf-variant-call-format-version-41) or Bed format (Personal Genome SNP format, http://genome.ucsc.edu/FAQ/FAQformat.html#format10), and allows specification of the background selection from two common sets of GWAS SNP typing microarrays. LD filtering is achieved at either r2 >= 0.8 or r2 >= 0.1 using 1000 genomes project population data. The outputs of the analysis are an interactive graphic for exploration of the analysis, a static pdf for printing or publication (Figure 1), and a table of enrichments in either an interactive or standard tab separated format.\n\nIn addition the code is available to download from https://github.com/iandunham/Forge, with the required database files available at ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/browser/forge. Installed on a Macbook Pro with core i7 processor, 16Gb RAM and a solid state hard drive, a typical FORGE analysis of a set of 55 SNPs with 1000 background tests is accomplished in around 30s, and in 35s with LD filtering.\n\nWe ran FORGE analysis on 260 phenotypes in the NCBI GWAS catalog NCBI GWAS catalog5,30 with a reported associated SNP count of 5 or more after LD pruning (see Supplementary Table S2 for list of phenotypes analysed and references) at genome-wide significance. Complete tables of results for all SNP sets analysed are included in the data directory of the Github release, https://github.com/iandunham/Forge. Thirty-five and 60 out of 260 SNP sets had at least one significant enrichment at the P value thresholds of 0.01 and 0.05 after correction for multiple testing, respectively (Table 1). A set of example positive outputs from this analysis is available from http://www.1000genomes.org/forge-gwas-catalog-example-gallery11 (PDF format). Removing SNPs that directly alter a protein coding exon from the GWAS catalog sets did not substantively alter the patterns of enrichments (data not shown).\n\nGWAS SNP set gives the phenotype of the study for which these SNPs were found to be associated as recorded in the GWAS catalog. SNP Count is the number of SNPs analysed before LD pruning. High and Low columns give the number of DNase1 cell samples found to be enriched for overlap in the FORGE analysis at binomial p >= 0.01 (High) and p >= 0.05 (Low) thresholds. Further details of the SNP sets analysed are given in Supplementary Table S2.\n\nFigure 1 shows a series of example FORGE analyses for autoimmune disease studies on the Roadmap Epigenome samples (references for the studies are provided in Supplementary Table S2). In each case there is a clear signal for enrichment of overlap with DNase I hotspots in the blood-derived samples including cells of immune function. In more detail, for those phenotypes where there is involvement of T cell activation or invasion in the aetiology (e.g. Crohn’s disease, Multiple sclerosis) there is enrichment in the CD3, CD4 and CD8 positive samples containing T cells as well as enrichment in the CD56 positive sample including NK cells. In addition, these disease SNPs overlap with hotspots present in the fetal thymus samples, consistent with the location of T cell maturation. Further signals specific to the individual aetiologies are also identified. Crohn’s disease SNPs show enrichment of overlap with hotspots in the fetal small and large intestine samples, as well as fibroblasts and skin cells. For inflammatory bowel disease SNPs there is a much more general enrichment, in addition to the specific immune cells, which may be consistent with the more generalized inflammation. In contrast, for autoimmune diseases where the primary involvement is a B cell response (Rheumatoid arthritis (RA), Systemic lupus erythematosus (SLE)), the most pronounced overlap enrichment is in CD19 positive samples characteristic of B cell activation or circulating plasma cells. In rheumatoid arthritis there is also some overlap enrichment for samples characteristic of T cells and thymocytes, but it is relatively less, and this is much less pronounced for SLE. Thus, FORGE analysis reveals tissue-specific enrichment of overlap for GWAS SNPs with regulatory regions indicative of known tissue involvement in the disease aetiology.\n\nThe tissue-specific enrichment of overlap is not specific to just autoimmune diseases (see results gallery at http://www.1000genomes.org/forge-gwas-catalog-example-gallery11). For instance, for QRS duration the GWAS associated SNPs are strongly enriched for overlap with fetal heart samples. GWAS SNPs associated with pulmonary function measured by spirometry are enriched for overlap with hotspots in fetal lung cells and lung cell lines. For red blood cell traits and platelet count the major overlap enrichment signal is in CD34 positive hematopoietic progenitor cells consistent with their role in both red blood cell and platelet development. In contrast for GWAS SNPs involved in height, the overlap enrichment is not tissue-specific but is more general over many tissues and cell lines. There are further examples displayed in the results gallery at http://www.1000genomes.org/forge-gwas-catalog-example-gallery11, in most cases consistent with expected disease aetiologies.\n\n\nDiscussion\n\nFORGE (Functional element Overlap analysis of the Results of GWAS Experiments) analysis is a straightforward and fast method to examine sets of nucleotide variants, typically identified in GWAS studies, for tissue-specific regulatory signals. It presents a graphical overview of overlap enrichment with DNase I hotspots that quickly provides evidence of a regulatory component to SNPs associated with a phenotype, and highlights potentially mechanistically relevant cells or tissues. A typical usage scenario would be to analyse a set of GWAS SNPs identified above genome wide significance to reveal the regulatory component of the association. Furthermore the cell or tissue enrichments might be consistent with prior expectation of the disease aetiology providing additional confidence in the SNP set identified, or might provide new insights as to potential sites of disease mechanism.\n\nThe statistical approach we used here relies on the careful matching of background behaviour of SNPs with calibrations by randomization for the per cell type enrichment. The underlying biases of GWAS SNP distributions with respect to TSS distance, maf and GC are not easy to model parametrically. However, other approaches which would make assumptions of homogeneity (such as the Poisson distribution) or of regional heterogeneity (Genome Structure Correction, 31) would not be able to capture these known biases. It is important to note that as alternate SNP resources are utilized in GWAS, the appropriate background SNPs must be used for control. For instance a switch to genotyping by genome sequencing or extensive use of imputation requires revision of the background. New and updated background sets can be implemented as required in particular for genome sequencing GWAS approaches and higher density genotyping arrays.\n\nNot all GWAS study SNP sets downloaded from the GWAS catalog showed overlap enrichment with the DNase I hotspots. In these cases all sample points were above the P value thresholds (blue points). This could occur because there is no regulatory component underlying the GWAS association in these phenotypes, because the associated SNPs do not contain mechanistically causal SNPs, because the relevant tissue is not present in the available DNase I datasets or because of low power in the GWAS study to detect regulatory effects. As further datasets are released by the ENCODE, Roadmap Epigenome and other projects, these can be incorporated into the database to provide coverage of further cell types. In addition, the approach could be readily extended to other data types including regions of specific histone modification as used by Trynka et al.23 or relevant transcription factor binding regions.\n\nSixty out of 260 sets of GWAS SNPs from the GWAS catalog for specific phenotypes had overlap enrichments detected in at least one DNase I hotspot sample (Table 1). As described above in several cases, the patterns of tissue-specific enrichment are highly evocative of the known aetiologies of the phenotypes, but can also reveal additional tissue involvements that require further investigation. We encourage interested parties to peruse the gallery of results for their own phenotypes, as well as running new SNP sets discovered in GWAS either through the web interface or with the standalone software.\n\n\nSoftware and data availability\n\nData of GWAS associated SNPs in regulatory regions37.\n\nFORGE is available through a web interface at http://browser.1000genomes.org/Homo_sapiens/UserData/Forge.\n\nThe source code for FORGE is available on GitHub at https://github.com/iandunham/Forge with the Forge.db sqlite database and background selection hash tables available at ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/browser/forge_11.\n\nFORGE has been successfully been installed and run on Mac OSX 10.8.4 and Red Hat Linux.\n\nhttps://github.com/iandunham/Forge\n\nhttps://github.com/F1000Research/Forge/releases/tag/v1.1\n\nhttp://dx.doi.org/10.5281/zenodo.1390038\n\nGNU GPL", "appendix": "Author contributions\n\n\n\nID and EB designed the analysis and wrote the paper. ID wrote software and ran analysis. SM conducted the clustering of cell types by DNase I regions. EK implemented the web interface. VI provided statistical advice and discussion.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by EMBL.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nMany thanks to Jan Quell for initial implementation of the pdf graphic and to Ramnath Vaidyanathan and @timelyportfolio for assistance with rCharts. We thank Prof Ajay Shah and Marc-Philip Hitz for comments on the results of cardiac phenotypes, and Graham Ritchie, and Nicole Soranzo for discussions.\n\n\nSupplementary Tables\n\n\n\n\nReferences\n\nVisscher PM, Brown MA, McCarthy MI, et al.: Five years of GWAS discovery. Am J Hum Genet. 2012; 90(1): 7–24. 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Reference Source\n\nBickel PJ, Boley N, Brown JB, et al.: Subsampling methods for genomic inference. Ann Appl Stat. 2010; 4(4): 1660–1697. Publisher Full Text\n\nAbecasis GR, Auton A, Brooks LD, et al.: 1000 Genomes Project Consortium, An integrated map of genetic variation from 1,092 human genomes. Nature. 2012; 491(7422): 56–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi H, Handsaker B, Wysoker A, et al.: The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009; 25(16): 2078–2079. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDjebali S, Davis CA, Merkel A, et al.: Landscape of transcription in human cells. Nature. 2012; 489(7414): 101–108. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarrow J, Frankish A, Gonzalez JM, et al.: GENCODE: the reference human genome annotation for The ENCODE Project. Genome Res. 2012; 22(9): 1760–1774. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNeph S, Kuehn MS, Reynolds AP, et al.: BEDOPS: high-performance genomic feature operations. Bioinformatics. 2012; 28(14): 1919–1920. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDunham I, Kulesha E, Iotchkova V, et al.: Data of GWAS associated SNPs in regulatory regions. figshare. 2015. Data Source\n\nDunham I, Herrero J: Forge: F1000Research/FORGE. Zenodo. 2015. Data Source" }	[ { "id": "7748", "date": "03 Mar 2015", "name": "Colin A. Semple", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThere is certainly demand for software to assess the functional impact of sequence variants within the noncoding majority of the genome, and the FORGE tool is intended to help address this. The choice of DNase I hotspots at a general indicator of regulatory activity is justified, and the interactive presentation of enrichment results in the web tool is useful. However, the manuscript gives the impression of having been put together in a hurry, arguably lacking the depth of exploration necessary to generate robust analyses or a sufficiently flexible tool. I make some specific suggestions for improvements below.Major revisionsThe authors adopt a sampling strategy to discover significant enrichments of a set of variants within datasets of annotated regulatory regions. Both the regulatory regions and the SNPs represented on genotyping platforms are not uniformly distributed across the genome, and show some degree of clustering. For example, both might be expected to be enriched in and around genes. It is not clear that the authors' sampling approach can generate a null distribution that faithfully represents this clustering, and there is therefore a danger that the significance of enrichments are exaggerated. Alternative approaches, such as Genome Structure Correction (cited by the authors) and circular permutation (eg Kindt et al, 2013, BMC Genomics 14:108) exist that could generate an appropriate null distribution, and the results could be compared to those from the sampling approach. As the authors point out in the Discussion, the results of these enrichment tests are dependent upon the background set used. For users to generate meaningful results they must select the appropriate genotyping platform as background. However, the web tool offers two background options, with the default as \"GWAS typing arrays\". Firstly, it is not clear what this option means, perhaps the union of SNPs for all NHGRI GWAS platforms? Secondly, given the notorious differences in ascertainment bias between genotyping platforms, this would seem to make it likely that most web tool users will use an inappropriate background (ie not matched to the platform they have used). Users employing the command line tool will face the task of tailoring each analysis to the appropriate background set, which may become labour intensive. This problem could be circumvented by using a general strategy (such as circular permutation) to produce a null distribution for each test. At the moment the DNAse datasets for different tissues are implicitly treated as independent, with each enrichment reported separately, which the authors admit is inaccurate. However, where the dependencies of these datasets are discussed the issue seems to be confused with controlling the false positive rate. The manuscript would benefit from a fuller discussion of this, and ideally some exploration of the interdependencies present eg using multiple regression I suspect the question most readers will be left with after reading this manuscript is, how does this tool relate to the others already available? If the intention is not to rigorously compare statistical methods, the authors could at least compare the features of other competing tools. The HaploReg tool (referenced by the authors) for example seems to include similar tests with more flexible LD calculations, and a greater range of functional annotation.Minor revisionsIt would probably make sense to order the enrichment results by the significance of enrichment, which does not appear to happen at the moment. A more flexible LD threshold for the web tool is desirable, at the moment only two values (0.1, 0.8) can be selected. The option of an FDR correction for the results would be a welcome addition to the Bonferroni correction implemented. Many journals now consider it unacceptable to use the comment \"data not shown\" to support assertions in the text, and this comment appears twice in this manuscript, along with one instance of \"results not shown\".", "responses": [] }, { "id": "9214", "date": "14 Jul 2015", "name": "Joanne Knight", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDunham et al. present a method to “identify significant cell specific enrichments in regulatory regions for sets of SNPs”. Results come with useful graphics. Major comment:Multiple testing is only controlled for across tissues and not across samples. Hierarchical clustering used to justify this but results are not given. I think these results would be useful.In light of this the authors should be more transparent about the expected false positive rate. In relation to figure 1, for 299 samples at the most stringent p-value (4x10-4) you would expect 1 sample to be designated significant just by chance in every two SNP sets tested. Given the empirically derived false positive rate of 0.5% per sample you would expect between one and two samples to be significant for each SNP set. Results would be similar in the case of those plotted in table 1 hence many would be likely to be false positives. When 260 SNP sets are analyzed further correction is required. Minor comments:It is not completely clear at what point the pruning was undertaken. In “Methods: GWAS SNP data:” it says only SNP sets with > 5 non-redundant SNPs were retained, but it listed pruning as a further step. Perhaps non-redundant refers to replicate SNPs but this needs to be clarified. In “Results: Gallery of Examples:” results with > 5 SNPs per set after LD pruning are discussed. There is some unnecessary repetition. For example the details in “Methods: Background SNP parameters” are repeated in too much detail in the results. Presumably Table 1 refers to both Roadmap and Encode data whereas figure 1 is only Roadmap. It would be helpful to clarify for table 1. The statement in the “Methods: FORGE analysis” section “The corrected thresholds are therefore more stringent than established by the random trials” is out of place as the random trials have not yet been explained. Furthermore it should be made clear that it is a false positive rate per sample and not per SNP set across all samples. In the “Results: FORGE analysis procedure” the enrichments identified greater than the threshold are considered positive, in the discussion points above the threshold are considered non-significant. This likely comes from using both the raw threshold and the -log10 of the threshold but should be clarified.", "responses": [] } ]	1	https://f1000research.com/articles/4-18
https://f1000research.com/articles/4-16/v1	16 Jan 15	{ "type": "Research Article", "title": "Ketamine infusion for patients receiving extracorporeal membrane oxygenation support: a case series", "authors": [ "Bethany Tellor", "Nicole Shin", "Thomas J. Graetz", "Michael S. Avidan", "Nicole Shin", "Thomas J. Graetz", "Michael S. Avidan" ], "abstract": "The use of ketamine infusion for sedation/analgesia in patients receiving extracorporeal membrane oxygenation (ECMO) therapy has not been described. The aims of this retrospective cohort study were to explore whether ketamine infusion for patients requiring ECMO therapy was associated with altered RASS scores, decreased concurrent sedative or opioid use, or with changes in vasopressor requirements. All patients on ECMO who received ketamine infusions in addition to sedative and/or opioid infusions between December 2013 and October 2014 at Barnes-Jewish Hospital in St. Louis were retrospectively identified. Patient characteristics and process of care data were collected.A total of 26 ECMO patients receiving ketamine infusion were identified. The median (inter quartile range [range]) age was 40 years (30-52 [25-66]) with 62% male. The median starting infusion rate of ketamine was 50 mg/hr (30-50 [6-150]) and it was continued for a median duration of 9 days (4-14 [0.2-21]). Prior to ketamine, 14/26 patients were receiving vasopressor infusions to maintain hemodynamic stability. Ketamine initiation was associated with a decrease in vasopressor requirement in 11/26 patients within two hours, and 0/26 required an increase (p<0.001). All patients were receiving sedative and/or opioid infusions at the time of ketamine initiation; 9/26 had a decrease in these infusions within two hours of ketamine initiation, and 1/26 had an increase (p=0.02; odds ratio for decrease to increase = 9; 95% CI, 1.14 to 71.04). The median (IQR[range]) RASS score 24 hours before ketamine initiation was -4 (-3 to -5, [0 to -5]) and after ketamine was -4 (-3 to -4 [-1 to -5]) (P = 0.614).Ketamine infusion can be used as an adjunctive sedative agent in patients receiving ECMO and may decrease concurrent sedative and/or opioid infusions without altering RASS scores. The hemodynamic effects of ketamine may provide the benefit of decreasing vasopressor requirements.", "keywords": [ "Ketamine", "Extracorporeal membrane oxygenation", "Intensive care unit", "Critically-ill", "Sedation" ], "content": "Introduction\n\nContinuous infusion of sedatives and opioids are commonly employed to alleviate pain, agitation, and anxiety in critically ill patients admitted to the intensive care unit1–2. Significant variability in sedation practices exist nationwide in the United States3, and efforts have been made to investigate systematic approaches to sedation management in order to improve patient outcomes4. As the adverse effects of over-sedation and prolonged sedation have become more apparent (e.g. longer duration of mechanical ventilation, longer ICU length of stay, increased mortality)5–7, the paradigm has shifted towards a “more is less” strategy8. Keeping critically ill patients less sedated and more interactive is supported by recent clinical practice guidelines on pain, agitation, and delirium9. However, severe agitation is common in critically ill patients and may have many underlying causes; agitation in such patients is associated with prolonged ventilator and ICU days as well as higher rates of self-extubation10. It is therefore important to determine the optimum level of sedation for every individual critically ill patient. Patients requiring extracorporeal membrane oxygenation (ECMO) therapy typically require sedation during the acute phase of their illness or when they are agitated such that they are at risk for self-harm, such as displacement of ECMO tubing. However, patients receiving ECMO support are challenging to sedate, often require escalating doses of sedatives and opioids to maintain an appropriate, safe level of sedation11. The challenges often relate to the dynamic alterations in pharmacokinetic (PK) and pharmacodynamic (PD) properties of commonly used sedatives and opioids in the setting of ECMO. Hemodilution, protein binding changes, changes in blood flow especially through the lungs, sequestration of medications by the ECMO circuit, binding of drugs to the oxygenator membrane, and organ function changes may impact the clinical effects of sedative and analgesic medications in this patient population10–19.\n\nKetamine is potentially a promising complementary sedative agent in the setting of ECMO in that it has hypnotic, analgesic, and amnesic properties owing to its activity at a variety of receptors. Ketamine is an antagonist of glutamate receptors as well as N-methyl-D-aspartate (NMDA) receptors and it has activity at all opioid receptors, though with different affinities and activities at those receptors. Ketamine also antagonizes nicotinic acetylcholine receptors noncompetitively and profoundly inhibits muscarinic receptors in addition to inhibiting L-type calcium channels20. Ketamine’s lack of respiratory depression, bronchodilating properties, in addition to generally being well tolerated hemodynamically (despite some myocardial depressant properties) theoretically make it an attractive choice for sedation and analgesia21. Recent clinical practice guidelines on pain, agitation, and delirium9 mention the use of ketamine as a possible adjunct for non-neuropathic pain management and suggest that nonbenzodiazepine sedatives such as propofol and dexmedetomidine may be preferred over benzodiazepine sedation. They do not provide recommendations for ketamine use as a continuous infusion. To our knowledge, no studies have compared clinical outcomes in ICU patients sedated with ketamine infusion to other agents. Ketamine may provide an alternative or adjunct option in difficult to sedate patients. As critical drug shortages continue to impact the selection of available drugs (e.g. propofol shortage22), it is important to review all relevant agents available for clinical use. The purpose of this study was to describe the use of ketamine infusion for patients receiving concurrent sedation/analgesia on ECMO support. The specific aims were to determine whether ketamine altered Richmond Agitation Sedation Scale (RASS) scores or decreased concurrent sedative, opioid, or vasopressor requirements.\n\n\nMethods\n\nThe Washington University School of Medicine Human Research Protection Office and the Protocol Review and Monitoring Committee approved this study (IRB # 201409142). This case series was conducted at Barnes-Jewish Hospital, a 1,250 bed urban teaching hospital affiliated with the Washington University School of Medicine in St. Louis, MO, between December 2013 and October 2014. All patients on ECMO support receiving ketamine infusion in addition to sedative and/or opioid infusions during the study period were retrospectively identified via an informatics query. There were no exclusion criteria. Patient characteristics, medical and surgical history, as well as process of care data were collected from electronic medical records. All ECMO patients are cared for in a single cardiothoracic intensive care unit. Doses and durations of all infusions of sedatives, opioids and vasopressor agents documented as administered during ketamine administration were collected. All available RASS scores charted 24 hours before and after ketamine initiation were collected on each patient. Statistical analysis was completed by using the SPSS software, version 18.0 (SPSS, Inc., Chicago, IL). Descriptive statistics were used to analyze the data collected. The median RASS scores as well as the median norepinephrine infusion rates (in patients who were receiving norepinephrine) before and after ketamine initiation were compared with a Wilcoxon signed-rank test. The McNemar test was used to assess whether or not a significant proportion of patients had a clinically meaningful increase or decrease in the dose of sedative, analgesic or vasopressor infusions two hours after ketamine initiation. Based on consensus of the investigators, the following minimum changes were arbitrarily pre-specified as clinically meaningful for sedative, analgesic and vasopressor infusions: propofol, 10 mcg/kg/min; midazolam, 1 mg/hour; dexmedetomidine, 0.2 mcg/kg/hour; fentanyl, 25 mcg/hour; norepinephrine 0.02 mcg/kg/min; and vasopressin 0.02 units/min.\n\n\nResults\n\nKetamine infusion for sedation and analgesia data for patients receiving extracorporeal membrane oxygenation support\n\nDataset 1_Patient demographics of patients receiving ketamine infusions while on extracorporeal membrane oxygenation therapy. Dataset 2_Continuous infusion details of patients receiving ketamine infusions while on extracorporeal membrane oxygenation therapy. Dataset 3_Vasopressor and sedative/opioid changes of patients receiving ketamine infusions while on extracorporeal membrane oxygenation therapy. Dataset 4_RASS scores before and after ketamine of patients receiving ketamine infusions while on extracorporeal membrane oxygenation therapy.\n\nClick here to access the data: 10.7910/DVN/28617\n\nA total of 26 ECMO patients receiving ketamine infusion were identified. The median (inter quartile range [range]) patient age was 40 years (30–52 [25–66]) with 62% male. The most frequent indication for ECMO support was respiratory failure associated with H1N1 influenza (46%). The median starting infusion rate of ketamine was 50 mg/hr (30–50 [6–150]) and it was continued for a median duration of 9 days (4–14 [0.2–21]). If the patient died in the hospital, that was considered the end of hospital stay. The median time from ECMO placement to ketamine initiation was 6 days (2–9 [0.9–22]). Nine patients (35%) died during their hospital stay. The most common causes of death were cardiopulmonary arrest and respiratory failure (Table 1).\n\nValues are expressed as n (%) unless specified otherwise.\n\nECMO = extracorporeal membrane oxygenation; VV = veno-venous; VA = veno-arterial; IQR = interquartile range\n\nOther = leukemia, Stiffman syndrome, cystic fibrosis, pneumonia\n\n£Cause of death documented in death summary note = cardiopulmonary arrest (2), respiratory failure (3), cardiogenic shock (2), acute respiratory distress syndrome (1), Hemophagocytic lymphohistiocytosis (1)\n\nAll patients were on a combination of one or more of the following agents: fentanyl, midazolam, propofol, and/or dexmedetomidine. At the time of ketamine initiation the median concurrent infusion rates of fentanyl (n=25), midazolam (n=19), propofol (n=10), and dexmedetomidine (n=9) were 200 mcg/hr (150–450 [50–900]), 7 mg/hr (4–9 [1–15]), 40 mcg/kg/min (23–48 [10–100]), and 1.2 mcg/kg/hr (1–1.4 [0.6–1.5]), respectively. The median starting infusion rate of ketamine was 50 mg/hr (30–50 [6–150]) and continued for a median duration of 9 days (4–14 [0.2–21]). Of note, 6 patients were receiving concurrent neuromuscular blocking agents during ketamine infusion (Table 2).\n\nValues are median (IQR, range), unless specified otherwise\n\nIQR = interquartile range.\n\nPrior to ketamine, 14/26 patients were receiving vasopressor infusions to maintain hemodynamic stability. Ketamine initiation was associated with a clinically meaningful decrease in vasopressor requirement in 11/26 patients within two hours, and 0/26 required an increase in this time period (p<0.001). In the 14 patients on norepinephrine infusions, the median (IQR [range]) dose before ketamine initiation was 0.1 mcg/kg/min (0.04–0.19 [0.02–0.26]) and two hours after ketamine initiation was 0.06 mcg/kg/min (0.01–0.11 [0–0.26]) (P=0.003). All patients were receiving sedative and/or opioid infusions at the time of ketamine initiation; 9/26 had a clinically meaningful decrease in these infusions within two hours of ketamine initiation, and 1/26 had an increase (p=0.02; odds ratio for decrease to increase = 9; 95% CI, 1.14 to 71.04) (Table 3). The median RASS score 24 hours before ketamine initiation was -4 (-3 to -5, [0 to -5]) and after ketamine was -4 (-3 to -4 [-1 to -5]) (P=0.614).\n\nBased on consensus of the investigators, the following minimum changes were arbitrarily pre-specified as clinically meaningful for sedative, analgesic and vasopressor infusions: propofol, 10 mcg/kg/min; midazolam, 1 mg/hour; dexmedetomidine, 0.2 mcg/kg/hour; fentanyl, 25 mcg/hour; norepinephrine 0.02 mcg/kg/min; and vasopressin 0.02 units/min.\n\n\nDiscussion\n\nECMO has repeatedly shown to complicate sedation management and optimization in the ICU as the PK/PD of sedatives and opioids become altered11,12,14–18. Lorazepam, midazolam, diazepam, propofol, and morphine have all been shown to be sequestered by ECMO circuits to some extent11,17,18, posing a challenge when faced with trying to provide appropriate sedation/analgesia. In an ex vivo study, lipophilic medications such as fentanyl and midazolam have been shown to sequester in the ECMO circuit resulting in significant loss, while morphine remained relatively stable18. A similar finding was shown by Shekar and collegaues11 who assessed sedation requirements in 30 patients receiving ECMO support. Significant increases in dosing requirements of midazolam and morphine were noted, with venovenous ECMO patients receiving higher sedative doses overall than patients on venoarterial ECMO. The effect of ECMO on the PK/PD and other drug properties of ketamine in patients on ECMO has not been studied.\n\nKetamine causes dissociation of the thalamus from the limbic cortex, resulting in patients resembling a cataleptic state. Patients may be unable to respond to sensory stimulation, may have nystagmus, and are able to conserve laryngeal and corneal reflexes21. Ketamine has been shown to decrease opioid consumption in surgical patients23,24, and is typically used as a one-time administration for analgesia, anesthesia induction, or procedural sedation. In addition to these indications, in some of the ICUs at our institution ketamine infusion has been added for patients who have been difficult to sedate to a target RASS. The use of ketamine infusion has particularly been adopted at our institution for patients requiring ECMO therapy, as these patients have anecdotally been difficult to sedate optimally. However, the safety and efficacy of ketamine for these patients has not been established.\n\nThe favorable hemodynamic and PK/PD profile that ketamine provides in patients not requiring ECMO therapy makes ketamine theoretically an attractive sedative agent generally for use in the ICU. The onset of action after IV administration occurs within 30 seconds, with a maximum effect in about 1 minute. The distribution half-life is 5–10 minutes while the elimination half-life is 2–3 hours, as ketamine undergoes extensive hepatic uptake and is primarily excreted in the urine21,25. There are no dosing adjustments provided by the manufacturer in the presence of hepatic or renal dysfunction, however the optimal dose and duration of ketamine infusion for sedation remains unknown. Interestingly, in recent years neuroprotection, immune modulating, and antidepressant properties have also been suggested for ketamine26–28.\n\nKetamine’s lack of respiratory depression, bronchodilation properties and ability to increase blood pressure, heart rate, and cardiac output (although it does act as a myocardial depressant) further add to the attractive profile of this agent21,29. Interestingly, a decrease in vasopressor requirements with ketamine initiation was observed in many of the patients in this series. Although charted RASS scores before and during ketamine infusion did not change, a decrease in sedatives and/or opioids within two hours of ketamine initiation was more common than an increase in sedatives and/or opioids.\n\nThis study has several limitations. The small sample size, lack of blinding and retrospective design limit our ability to make causal inferences. Although the majority of patients receiving vasopressors had a meaningful decrease in their requirements, we cannot exclude the fact that a decrease in vasopressors may have occurred unrelated to ketamine infusion. The observational design also limits our ability to determine why certain drugs were chosen concurrently or why one was decreased prior to another. Although RASS values were collected, we were unable to determine the specified RASS goal for each patient. A RASS goal of 0 to -2 is targeted in most ICU patients, however 6 patients in this series were receiving neuromuscular blocking agents, rendering RASS values uninterpretable, while other patients received escalating doses of sedatives/opioids despite RASS values < -2. Furthermore, the use of a RASS score to monitor and guide sedation with ketamine has not been validated.\n\nTo our knowledge, this is the first study evaluating the use of ketamine infusion in patients on ECMO support. This study demonstrated that ketamine infusion can be used as an adjunctive agent in patients receiving ECMO with possible sedative/opioid sparing effects. The cardiovascular properties may provide the hemodynamic benefit of reducing vasopressor requirements. The lack of understanding related to optimizing pharmacotherapy in patients on ECMO has led to ongoing research, aimed at improving patient care13,19,30. All appropriate drugs should be considered in the absence of clear standards or guidelines for analgesia/sedation in patients receiving ECMO support. More rigorous study is warranted to further investigate the suitability of ketamine for sedation in patients receiving ECMO support.\n\n\nData availability\n\nDataverse: Dataset 1. Ketamine infusion for sedation and analgesia data for patients receiving extracorporeal membrane oxygenation support, doi: 10.7910/DVN/2861731", "appendix": "Author contributions\n\n\n\nAll authors contributed to this research and preparation of the manuscript. All authors agreed with the final content. Tellor: conceptualization, IRB approval, data acquisition, data analysis, manuscript preparation, manuscript revision. Shin: data acquisition, data analysis, manuscript preparation. Graetz: conceptualization, manuscript preparation. Avidan: conceptualization, data analysis, manuscript preparation, manuscript revision.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in the funding of this work.\n\n\nReferences\n\nPayen JF, Chanques G, Mantz J, et al.: Current practices in sedation and analgesia for mechanically ventilated critically ill patients: a prospective multicenter patient-based study. Anesthesiology. 2007; 106(4): 687–95. PubMed Abstract \| Publisher Full Text\n\nPatel SB, Kress JP: Sedation and analgesia in the mechanically ventilated patient. Am J Respir Crit Care Med. 2012; 185(5): 486–497. PubMed Abstract \| Publisher Full Text\n\nRhoney DH, Murry KR: National survey of the use of sedating drugs, neuromuscular blocking agents, and reversal agents in the intensive care unit. J Intensive Care Med. 2003; 18(3): 139–45. PubMed Abstract \| Publisher Full Text\n\nJackson DL, Proudfoot CW, Cann KF, et al.: A systematic review of the impact of sedation practice in the ICU on resource use, costs and patient safety. Crit Care. 2010; 14(2): R59. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShehabi Y, Bellomo R, Reade MC, et al.: Sedation Practice in Intensive Care Evaluation (SPICE) Study Investigators; ANZICS Clinical Trials Group: Early intensive care sedation predicts long-term mortality inventilated critically ill patients. Am J Respir Crit Care Med. 2012; 186(5): 724–731. PubMed Abstract \| Publisher Full Text\n\nShehabi Y, Chan L, Kadiman S, et al.: Sedation Practice in Intensive Care Evaluation (SPICE) Study Group investigators:Sedation depth and long-term mortality in mechanically ventilated critically ill adults: a prospective longitudinal multicentre cohort study. Intensive Care Med. 2013; 39(5): 910–918. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKollef MH, Levy NT, Ahrens TS, et al.: The use of continuous i.v. sedation is associated with prolongation of mechanical ventilation. Chest. 1998; 114(2): 541–8. PubMed Abstract \| Publisher Full Text\n\nPeitz GJ, Balas MC, Olsen KM, et al.: Top 10 myths regarding sedation and delirium in the ICU. Crit Care Med. 2013; 41(9 Suppl 1): S46–56. PubMed Abstract \| Publisher Full Text\n\nBarr J, Fraser GL, Puntillo K, et al.: Clinical practice guidelines for the management of pain, agitation, and delirium in adult patients in the intensive care unit. Crit Care Med. 2013; 41(1): 263–306. PubMed Abstract \| Publisher Full Text\n\nWoods JC, Mion LC, Connor JT, et al.: Severe agitation among ventilated medical intensive care unit patients: frequency, characteristics and outcomes. Intensive Care Med. 2004; 30(6): 1066–1072. PubMed Abstract \| Publisher Full Text\n\nShekar K, Roberts JA, Mullany DV, et al.: Increased sedation requirements in patients receiving extracorporeal membrane oxygenation for respiratory and cardio-respiratory failure. Anaesth Intensive Care. 2012; 40(4): 648–655. PubMed Abstract\n\nShekar K, Fraser JF, Smith MT, et al.: Pharmacokinetic changes in patients receiving extracorporeal membrane oxygenation. J Crit Care. 2012; 27(6): 741.e9–741.e18. PubMed Abstract \| Publisher Full Text\n\nShekar K, Roberts JA, Smith MT, et al.: The ECMO PK Project: an incremental research approach to advance understanding of the pharmacokinetic alterations and improve patient outcomes during extracorporeal membrane oxygenation. BMC Anesthesiol. 2013; 13: 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAhsman MJ, Hanekamp M, Wildschut ED, et al.: Population pharmacokinetics of midazolam and its metabolites during venoarterial extracorporeal membrane oxygenation in neonates. Clin Pharmacokinet. 2010; 49(6): 407–419. PubMed Abstract \| Publisher Full Text\n\nDagan O, Klein J, Bohn D, et al.: Effects of extracorporeal membrane oxygenation on morphine pharmacokinetics in infants. Crit Care Med. 1994; 22(7): 1099–1101. PubMed Abstract \| Publisher Full Text\n\nMulla H, Lawson G, Peek GJ, et al.: Plasma concentrations of midazolam in neonates receiving extracorporeal membrane oxygenation. ASAIO J. 2003; 49(1): 41–47. PubMed Abstract\n\nMulla H, Lawson G, von Anrep C, et al.: In vitro evaluation of sedative drug losses during extracorporeal membrane oxygenation. Perfusion. 2000; 15(1): 21–26. PubMed Abstract \| Publisher Full Text\n\nShekar K, Roberts JA, Mcdonald CI, et al.: Sequestration of drugs in the circuit may lead to therapeutic failure during extracorporeal membrane oxygenation. Crit Care. 2012; 16(5): R194. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShekar K, Roberts JA, Welch S, et al.: ASAP ECMO: Antibiotic, Sedative and Analgesic Pharmocokinetics during Extracorporeal Membrane Oxygenation: a multi-centre study to optimize during therapy during ECMO. BMC Anesthesiol. 2012; 12: 29. Publisher Full Text\n\nSinner B, Graf BM: Ketamine. Handbook of experimental pharmacology. 2008; 182: 313–33. Publisher Full Text\n\nParashchanka A, Schelfout S, Coppens M: Role of novel drugs in sedation outside the operating room: dexmedetomidine, ketamine and remifentanil. Curr Opinion Anesthesiol. 2014; 27(4): 442–7. PubMed Abstract \| Publisher Full Text\n\nJensen V, Rappaport BA: The reality of drug shortages--the case of the injectable agent propofol. N Engl J Med. 2010; 363(9): 806–07. PubMed Abstract \| Publisher Full Text\n\nDe Pinto M, Jelacic J, Edwards WT: Very-low-dose ketamine for the management of pain and sedation in the ICU. J Opioid Manag. 2008; 4(1): 54–6. PubMed Abstract \| Publisher Full Text\n\nZakine J, Samarcq D, Lorne E, et al.: Postoperative ketamine administration decreases morphine consumption in major abdominal surgery: a prospective, randomized, double-blind, controlled study. Anesth Analg. 2008; 106(6): 1856–61. PubMed Abstract \| Publisher Full Text\n\nKetamine hydrochloride injection [prescribing information]. Lake Forest, IL: Hospira Inc; January 2013. Reference Source\n\nHudetz JA, Pagel PS: Neuroprotection by ketamine: a review of the experimental and clinical evidence. J Cardiothorac Vasc Anesth. 2010; 24(1): 131–42. PubMed Abstract \| Publisher Full Text\n\nMurrough JW: Ketamine as a novel antidepressant: from synapse to behavior. Clin Pharmacol Ther. 2012; 91(2): 303–9. PubMed Abstract \| Free Full Text\n\nMathews DC, Zarate CA: Current status of ketamine and related compounds for depression. J Clin Psychiatry. 2013; 74(5): 516–517. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiller AC, Jamin CT, Elamin EM: Continuous intravenous infusion of ketamine for maintenance sedation. Minerva Anestesiol. 2011; 77(8): 812–820. PubMed Abstract\n\nColumbia University, Ketamine and Extracorporeal Membrane Oxygenation (ECMO). In: ClinicalTrials.gov [cited 2014 Dec 18]. Reference Source\n\nTellor B, Shin N, Graetz TJ, et al.: Ketamine infusion for sedation and analgesia data for patients receiving extracorporeal membrane oxygenation support. Dataverse. 2014. Data Source" }	[ { "id": "7693", "date": "18 Feb 2015", "name": "Kevin Thornton", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article is a retrospective case series reviewing the use of ketamine as an adjunctive sedative agent for patients requiring ECMO support due to cardiopulmonary failure. The authors reviewed patient characteristics including concurrent sedative, analgesic, and vasopressor infusion requirements as well as RASS scores both before and after initiation of ketamine infusion. The authors defined what they considered to be a 'clinically significant' changes in the doses of several agents including fentanyl, midazolam, propofol, and norepinephrine. While the majority of patients had a 'clinically significant' decrease in the doses of both vasopressors and other sedative/analgesic agents, these changes were still quite small. The authors rightly note that they are not able to make a causal associations given the retrospective nature of their data, but their inferences are sound based on pharmacologic and physiologic principles. This case series demonstrates that ketamine appears to be an adjunctive agent that can safely be used in patients on ECMO and may have the benefit of decreasing the requirements for other sedatives and vasopressors, as well. This article is well-written and this topic warrants further investigation in a prospective manner.", "responses": [] }, { "id": "7631", "date": "05 Mar 2015", "name": "Anthony R. Absalom", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary:The authors should be congratulated on an excellent article reporting an analysis of routinely collected clinical data. They have addressed a clinically relevant subject and with an important research question and a plausible and intuitive underlying hypothesis. Although retrospective, the study methodology is good and the statistical analysis is appropriate. The results are clear, although the manner of presentation could be improved. The discussion is informative and educational. The study limitations are appropriately recognised and discussed, and suggestions for future investigation are made. The conclusions are well supported by the data. Abstract Clear and concise. It describes the background, results and the conclusions accurately.IntroductionThe introduction provides a clear and concise summary of the clinical background and relevance of the study. The proposed hypothesis is clinically very relevant and plausible. The specific aims of the study are stated clearly.MethodsThe description of the methods is completely clear. Appropriate methodology was applied. To assess the influence of ketamine on sedative and vasopressor requirements, the authors arbitrarily chose, by consensus, definitions of “ clinically meaningful minimal changes in medication after the start of ketamine infusion”. This is a reasonable approach. The statistical analysis is clearly described and is appropriate. ResultsWe have two comments about the presentation of the results. The first is that table 2 is somewhat confusing. It is not clear what the “maximum” and “minimum” infusion rates refer to. We suggest that the authors clearly state the time period over which these maximum and minimum infusion rates were determined. If these rates are not the rates after starting ketamine, then we suggest that the authors present the median (IQR, range) infusion rates during some (arbitrary) time period after starting ketamine.Our second comment about the results concerns the consistency of units used in presentation of sedative and vasopressor infusion rates. In the methods the authors state arbitrarily chosen definitions of clinically meaningful changes in medication. These thresholds are defined in mcg/kg/min units for each of the drugs. In the text and in table 2 of the results, the infusion rates of fentanyl and midazolam are reported in different units (mcg/min). Thereafter the authors only report the numbers of patients in which there was a clinically relevant change. To more easily give the reader an idea of the magnitude of the relative changes in infusion rate of fentanyl and midazolam, we suggest that for these drugs the before and ketamine infusion rates are also presented in mcg/kg/min.DiscussionDiscussion is very well written and informative. The study limitations are well recognised. The conclusions are supported by the data.ReferencesAppropriate use of references", "responses": [] } ]	1	https://f1000research.com/articles/4-16
https://f1000research.com/articles/4-13/v1	15 Jan 15	{ "type": "Case Report", "title": "Case Report: Persistent erectile dysfunction in a man with prolactinoma", "authors": [ "Justin Badal", "Ranjith Ramasamy", "Tariq Hakky", "Aravind Chandrashekar", "Larry Lipshultz", "Justin Badal", "Tariq Hakky", "Aravind Chandrashekar" ], "abstract": "Erectile dysfunction has been explored as a condition secondary to elevated prolactin; however, the mechanisms by which elevated prolactin levels cause erectile dysfunction have not yet been clearly established. We here present a patient with a history of prolactinoma who suffered from persistent erectile dysfunction despite testosterone supplementation and pharmacological and surgical treatment for the prolactinoma. Patients who have had both prolactinemia and erectile dysfunction have been reported in the literature, but we find no report of a patient with persistent erectile dysfunction in the setting of testosterone supplementation and persistent hyperprolactinemia refractory to treatment. This case provides evidence supporting the idea that suppression of erectile function occurs in both the central and peripheral nervous systems independent of the hypothalamic-pituitary-gonadal axis.", "keywords": [ "prolactin", "sexual dysfunction" ], "content": "Introduction\n\nProlactinomas are the most common type of pituitary adenoma and account for 30% of all clinically recognized cases of pituitary adenomas1. Generally, prolactinomas arise in the second to fourth decade of life and are more quickly recognized in women than in men because the women experience an abrupt cessation of menses1–3. In men, hyperprolactinemia (HPRL) causes hypogonadotropic hypogonadism leading to decreased libido, impotence, infertility, gynecomastia or galactorrhea1,3,4. This effect is due to prolactin’s inhibitory action on gonadotropin releasing hormone, which ultimately results in decreased luteinizing hormone levels and decreased testosterone production by the testes1,2,4.\n\n\nCase report\n\nThe patient is a 23-year-old male who was seen initially in our clinic because of bilateral nipple discharge. His past medical history included a prolactinoma initially diagnosed when, at the age of 15, he reported changes in his vision that were worst on the right side, with intermittent complete darkening. He was subsequently found to have a pituitary macroadenoma on imaging and a prolactin level of approximately 4000 ng/ml. After therapy with cabergoline 0.5mg, 3 times per week, his prolactin decreased to 30–40 ng/ml, but there was no increase in testosterone. He was given supplemental testosterone therapy with testosterone cypionate. Three years after initial diagnosis, his prolactin levels had increased to 1500 ng/ml, despite therapy with cabergoline.\n\nHe underwent transphenoidal resection 4 years after diagnosis (age 19) with removal of the sellar bulk of tumor, but with residual right cavernous tumor seen on post operative imaging. The prolactin levels decreased from 1500 to 300 ng/ml, but continued to rise over the next few months despite therapy with cabergoline. Because of worsening symptoms a year later (age 20), he had gamma knife therapy of the residual right cavernous portion. However, prolactin remained elevated in the 500 – 600 ng/ml range, even though he was receiving cabergoline.\n\nWhen he came to our clinic, he could not achieve an erection sufficient for masturbation or sexual activity, and he did not have nocturnal erections. With adequate testosterone supplementation and anastrazole therapy, he experienced increased energy levels and elevated libido and has expressed the desire for treatment for his erectile dysfunction.\n\n\nClinical findings\n\nOn physical examination, the patient was well developed and well nourished. His external chest exam was significant for gynecomastia despite recent bilateral mastectomy. The testes were 12cc in volume.\n\n\nDiagnostic assessment\n\nHormone testing revealed a prolactin level of 300 ng/ml (normal range is 3.0 – 30.0 ng/ml) while he was receiving therapy with cabergoline. Luteinizing and Follicle Stimulating Hormone remained low at all points during treatment ranging between 0.00 to 0.22 mIU/ml (normal range, 1.2 – 7.8 mIU/ml) and 0.09 to 0.36 mIU/ml (normal range, 1.3 to 11.4 mIU/ml), respectively. The patient’s testosterone ranged from 384 – 1600 ng/dl (normal range of 200 – 1000 ng/dl) while receiving testosterone supplementation with testosterone cypionate (200mg IM injection once a week initiated at the initial clinic visit).\n\n\nTherapeutic intervention\n\nA duplex penile ultrasound following injection of 0.30cc of TriMix demonstrated some engorgement of the patient’s penis, with forty percent rigidity and no venous leak. Peak systolic velocity and end diastolic velocity were within normal limits. Despite treatment with tadalafil 5mg daily, he did not notice improvement in erectile function. He is now using a vacuum erection device for sexual activity. He was offered several different methods for improving his erectile function, including intracavernosal injections and an inflatable penile prosthesis and is currently debating which option to choose. Ultimately, the patient desires a more permanent solution to his erectile dysfunction, having stated that he is generally not satisfied with his current medical management for the erectile dysfunction. He notes that he is otherwise very pleased with the effects of the testosterone supplementation.\n\n\nDiscussion\n\nIn 2013, a published case study described a man with a pituitary adenoma who reported loss of libido and inability to have an erection over a period of 8 years5. All results of routine laboratory tests, including serum testosterone levels, were normal. Contrast MRI demonstrated a homogenous enhancement of the pituitary, and the patient was found to have a significantly elevated prolactin level. Despite normal testosterone levels, he had significant erectile dysfunction. After treatment with cabergoline, the patient achieved normal erectile function and improvement in neurologic symptoms. Similar to our patient, this patient had erectile dysfunction and hyperprolactinemia despite normal levels of testosterone, suggesting that prolactin has different ways of exerting its effect on erectile function.\n\nThe relationship between hyperprolactinemia and testosterone was first described in 1978 when a group of hypogonadal men with hyperprolactinemia were reported to have regained their sexual function only after receiving bromocriptine therapy, even though they had previously received adequate amounts of supplemental testosterone6 (Figure 1). Another study in 2004 evaluated the effect of cabergoline on men with hyperprolactinemia and erectile dysfunction. The study measured episodes of nocturnal penile tumescence (NPT) and found that when hyperprolactinemic hypogonadic men were treated with cabergoline, the number of monitored erections during sleep (NPT) increased7.\n\nGonadotropin releasing hormone (GnRH) stimulates release of Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) from the anterior pituitary which stimulates Leydig cells and Sertoli cells in the production of testosterone and sperm, respectively. Inhibin, produced by the Sertoli cells, negatively feeds back on FSH release from the anterior pituitary. Testosterone negatively feeds back on GnRH release from the hypothalamus and LH from the anterior pituitary, self-regulating its levels. In the dashed blue box, elevated prolactin secretion caused by a prolactinoma leads to pathologic inhibition of GnRH release from the hypothalamus and downstream inhibition of testosterone synthesis.\n\nUnfortunately, there has been a misunderstanding of the hypothalamic-pituitary-gonadal axis with regard to erectile function and prolactin. Elevated levels of prolactin lead to an impairment of the pulsatile release of luteinizing hormone resulting in decreased serum testosterone secretion. This hypogonadism was generally accepted as the main cause of erectile dysfunction. However, as we observed, despite adequate supplementation of testosterone as initial therapy for erectile dysfunction, the patient continued to be unable to have an erection. This mixed picture of elevated prolactin and “normal” testosterone raises the question of where prolactin is exhibiting its inhibitory effect.\n\nProlactin exerts its effects in a variety of locations, including the hypothalamus and the cavernosal bodies8. Hyperprolactinemia leads to increased expression of tyrosine hydroxylase mRNA in regions of the hypothalamus associated with sexual and erectile function. The fact that prolactin regulates the synthesis, release, and turnover of dopamine in hypothalamic neurons explains the initial increase in libido and erectile function observed in a study of male rats with acute hyperprolactinemia; however, a subsequent decrease in erectile function was observed as prolactin levels remained elevated, suggesting a down regulation of dopamine receptors secondary to chronically elevated levels of prolactin (Figure 2). This potential dysregulation of dopamine could explain the central inhibitory effect of hyperprolactinemia on erectile function, especially since erectile function has been shown to be corrected following bromocriptine administration before hypogonadism has been adequately treated. This evidence points to prolactin exerting its effect outside of the hypothalamic-pituitary-gonadal axis and acting independently of depressed GnRH, LH, or testosterone levels.\n\nHypothalamic neurons are stimulated by chronically elevated levels of prolactin to excessively release dopamine which results in dopamine receptor internalization and dysregulation of the downstream signal for sexual function.\n\nOur patient had experienced a prolonged course of hyperprolactinemia and, with indicated treatment, did not adequately recover erectile function. Although the testosterone supplementation resulted in resolution of his other hypogonadal issues – namely loss of energy and mood swings – the elevated prolactin may have severely altered the excitatory pathways in the hypothalamus, necessitating longer term or more invasive treatment to ameliorate the offending suppression and erectile dysfunction.\n\n\nConsent\n\nWritten informed consent for publication of clinical details was obtained from the patient.", "appendix": "Author contributions\n\n\n\nJB wrote the draft of the manuscript. RR conceived the idea. TH and AC managed the patient and followed up with the patient. LIL provided overall supervision.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nRanjith Ramasamy is a K12 Scholar supported by a Male Reproductive Health Research Career Development Physician-Scientist Award (HD073917- 01) given by National institute of child health and development (NICHD)\n\n\nReferences\n\nKumar V, Abbas AK, Fausto N, et al.: Robbins & Cotran Pathologic Basis of Disease, 8th Edition. Philadelphia: Saunders. 2009; 1103–4. Reference Source\n\nRogers A, Karavitaki N, Wass JA: Diagnosis and management of prolactinomas and non-functioning pituitary adenomas. BMJ. 2014; 349: g5390. PubMed Abstract \| Publisher Full Text\n\nAlmalki MH, Buhary B, Alzahrani S, et al.: Giant prolactinomas: clinical manifestations and outcomes of 16 Arab cases. Pituitary. 2014. PubMed Abstract \| Publisher Full Text\n\nRomijn JA: Chapter 13 - Hyperprolactinemia and prolactinoma. In: Eric Fliers MK and JAR, editor. Handbook Clin Neurol. Elsevier. 2014; 124: 185–95. PubMed Abstract \| Publisher Full Text\n\nAnand KS, Dhikav V: Hyperprolactinemia: an unusual cause of erectile dysfunction. Arch Sex Behav. 2013; 42(3): 341. PubMed Abstract \| Publisher Full Text\n\nCarter JN, Tyson JE, Tolis G, et al.: Prolactin-screening tumors and hypogonadism in 22 men. N Engl J Med. 1978; 299(16): 847–52. PubMed Abstract \| Publisher Full Text\n\nDe Rosa M, Zarrilli S, Vitale G, et al.: Six months of treatment with cabergoline restores sexual potency in hyperprolactinemic males: an open longitudinal study monitoring nocturnal penile tumescence. J Clin Endocrinol Metab. 2004; 89(2): 621–5. PubMed Abstract \| Publisher Full Text\n\nWalia R, Bhansali A, Dutta P, et al.: Recovery pattern of hypothalamo-pituitary-testicular axis in patients with macroprolactinomas after treatment with cabergoline. Indian J Med Res. 2011; 134(3): 314–9. PubMed Abstract \| Free Full Text" }	[ { "id": "7330", "date": "29 Jan 2015", "name": "Nelson Bennett", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting case report involving a young man with persistent erectile dysfunction in the setting of a prolactinoma. Introduction:What are some of the actions of prolactin in normal male physiology?Case Report:The word \"worst\" is used inappropriately.Why was a 15 year old male started on testosterone cypionate instead of HCG or other medications that would not compromise his fertility?Are there plans for continued treatment of the prolactinoma? Clinical Findings:Please greatly expand this section. What are his vital signs, visual acuity, body habitus, body hair distribution, tanner stage, penile size, etc?Diagnostic Assesment:Aside from the prolactin, LH, FSH, and Testosterone levels, what is his TSH level and hematocrit level? Therapeutic Intervention:Was the duplex penile U/S completed in a multidosing regimen?What were the actual duplex parameters..i.e. the PSV, EDV, and RI?Discussion:No issues, however, the integrity of the manuscript would be enhanced if the authors would offer suggestions on how to assess and treat these complex patients.", "responses": [] }, { "id": "7689", "date": "25 Feb 2015", "name": "Wayland Hsiao", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an excellent case report that raises the idea that prolactin may have other mechanisms of action for suppression of erectile function besides the most commonly cited reason of low testosterone.This case highlights the idea that there are certainly multiple mechanisms by which any biological alteration (high prolactin) can affect normal physiology (erectile function).I would hope that the authors will comment on whether there are any plans to further intervene in this young man's prolactin beyond the already mentioned therapies. Also, for the evaluation of erectile function, more information could be given on the specific hemodynamic parameters obtained as well as any mention of what kind of baseline sexual function he has (either an IIEF score or other standardized questionaire).However, overall, a great report to highlight that we don't understand all perturbation that go one when hormones are abnormal, and the \"traditional\" thinking of single pathways is probably folly and more a representation of reading about a disease in a textbook rather than critically thinking about the pathophysiology and the limitations of our current medical knowledge.", "responses": [] }, { "id": "7928", "date": "12 Mar 2015", "name": "Ryan Smith", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have composed an excellent case report highlighting the fact that multiple mechanisms can impair erectile function. In addition, hormone replacement is not typically sufficient to resolve this issue. The pathophysiology behind the inhibitory effect of prolactin appears to extend beyond hypogonadism and is intriguing.1. Do the authors have any IIEF/SHIM data at baseline and follow-up?2. Perhaps the authors can comment on the use of TST as opposed to alternative agents (HCG) which would not impact fertility.3. I would add some additional comments regarding the physical exam characteristics (i.e. secondary sex characteristics, etc.)4. Do the authors have any conjecture as to the mechanisms underlying the impact of hyperprolactinemia on this patients ED despite TST?", "responses": [] } ]	1	https://f1000research.com/articles/4-13
https://f1000research.com/articles/4-11/v1	14 Jan 15	{ "type": "Research Note", "title": "A new record of Actinobacteria isolated from soil in Jerusalem and their enzymatic potential", "authors": [ "Samira R. Mansour", "Ahmed M. Abdel-Azeem", "Samy Salem Soliman Abo-Deraz", "Ahmed M. Abdel-Azeem", "Samy Salem Soliman Abo-Deraz" ], "abstract": "Actinobacteria are well recognized for their bioactive compounds.They are considered as a promising source of wide range of important enzymes, some of which are produced on an industrial scale. In this study, 35 isolates of actinomycetes were isolated from soil samples collected in the area of Al-Aqsa Mosque in Jerusalem, Israel. To our knowledge, this is the first study of actinomycetes from this terrestrial environment. The efficiency of the isolated actinobacteria in the production of amylase, cellulase, protease, tyrosinase, lipase, catalase and phosphatase was studied. Isolates obtained showed some activity and other completely failed to produce such enzymes. From total 35 isolates, only three isolates (8.6%) showed ability to produce protease, four isolates (11.4%) for lipase, five isolates (14.3%) for tyrosinase and two isolates (5.7%) for phosphatase enzymes. However, all isolates were positive for amylase and catalase enzymes; vice versa for cellulase enzyme all isolates failed to degrade cellulose in the form of carboxymethylcellulose.", "keywords": [ "Actinobacteria", "Biotechnology", "Natural products", "Enzyme production", "Jerusalem." ], "content": "Introduction\n\nActinobacteria represent one of the most diverse groups of filamentous bacteria capable of surviving in a number of ecological niches due to their bioactive potential. They are representative of terrestrial microorganisms and usually are isolated from soils. Actinobacteria have gained special importance as the most potent source of antibiotics (Kandasamy et al., 2012) and other bioactive secondary metabolites (Solecka et al., 2012). Their metabolic potential offers a strong area of research. Accordingly, the role of actinomycetes in biotechnology and medicine is well known and these industries are always looking for novelty bioactive compounds. While most of the studies on actinobacteria have focused on antibiotic production, only few reports have focused on their enzymatic potential.\n\nActinobacteria are considered as a promising source of a wide range of enzymes. Some of them are produced on an industrial scale, but many other remained to be harnessed. The bacteria have the ability to degrade a wide range of hydrocarbons, pesticides, and aliphatic and aromatic compounds (Sambasiva Rao et al., 2012). They perform microbial transformations of organic compounds, a field of great commercial value. Members of many genera of actinobacteria have potential for use in the bioconversion of underutilized agricultural and urban wastes into high-value chemical products (Crawford, 1988). Some actinobacteria secrete enzymes responsible for the degradation of lignocelluloses in lignin, cellulose and hemicellulase, others may secrete enzymes that can only partially achieve this breakdown (Mason et al., 2001). Here the purpose of this preliminary study was to isolate and screen new actinobacterial isolates for their ability to degrade organic compounds via the secretion of enzymes like amylase, cellulase, protease, tyrosinase, lipase, catalase, and phosphatase. Due to the high disturbance level of biodeterioration occurred in the area of Al-Aqsa mosque in Jerusalem, Israel, the soil specimens from such place may contain novel actinobacterial colonies. Meanwhile, to the author’s knowledge, no previous studies concerned organisms involved in biodeterioration in this location.\n\n\nMaterials and methods\n\nSoil samples were collected 5 cm below the soil surface from two different sites at Northern part of Al-Aqsa mosque in Jerusalem. The soils collected from the area around Al-Aqsa are characterized by high pH ranged from 8.15 to 8.32 with organic carbon 9.61% – 12.28%. Soil textures were clay and clay loam. No attempts have been made before to isolate actinomycetes from these areas. The soil samples collected were subjected to sieving to remove plant debris and were then pre-treated by drying in open air for 2 days. Samples of 5 g were mixed with 50 ml of sterile saline solution (0.85% NaCl) and incubated at room temperature (27±2°C) for 1 hour on orbital shaker with vigorous shaking. Soil suspension was subjected to serial dilutions and then pipetted and spread onto starch casein medium (SCM, with the following ingredients gm-1Le: casein powder 1.0, starch 10.0, sodium nitrate, 3.0; agar 15.0; final pH 7.2±0.2) supplemented with antifungal cycloheximide (50 mg-1L) as described by Mansour (2003). After 7 days of incubation at 30°C, actinomycete colonies were picked up and purified onto SCM medium using streak plate techniques. The pure colonies of actinomycetes were subcultured onto starch casein slants and incubated for 3–7 days at 30°C.\n\nPurified actinobacterial isolates were identified to genus level using different tools, morphological, cultural and physiological characteristics following the standard techniques as presented in Bergey’s Manual of Systematic Bacteriology (Anon, 1989). For morphological characterization, sterile slide oblique technique was applied (Mansour, 2003). Actinomycete colonies were streaked onto SCM, where the slide was inserted in the agar plate with an angle of 45° and incubated at 30°C for 3 and 7 days. After each incubation period, the growth of actinomycetes was examined taking the slides out from the agar and staining the actinobacteria growth using Gram stain. The slides were then examined using a light microscope (Leica, Model DMLB). Spore orientation and their morphological types were examined. Culture characterization was carried out using different culture media: SCM (Kuster, 1959), glycerol asparagine agar (reagents g-1L: L-asparagine, 1.0; dipotassium phosphate, 1.0; trace salt solution (ml) 1.0; agar 20.0; 1ml of trace salt solution contains, ferrous sulfate heptahydrate, 0.001; manganese chloride tetrahydrate, 0.001; zinc sulfate, heptahydrate 0.001; final pH 7.4±0.2 (Pridham & Lyons, 1961), glucose asparagine agar (reagents g-1L: glucose, 10.0; asparagine, 0.5; di-potassium hydrogen phosphate, 0.5; agar, 15.0; pH 7.4 (Waksman, 1961), yeast extract-malt extract agar (reagents g-1L: yeast extract, 3.0; malt extract, 3.0; dextrose, 4.0; agar, 20.0; pH 7.2 (Pridham et al., 1956), inorganic salt-starch agar (reagents g-1L: soluble starch, 10.0; di-potassium hydrogen phosphate, 1.0; magnesium sulfate, 1.0; sodium chloride, 1.0; ammonium sulfate, 2.0; calcium carbonate, 2.0; trace slats solution, 1 ml; agar, 15.0 (Kuster, 1959) and oat meal agar (reagents g-1L: oat meal, 2.0 and agar, 15.0 (Kuster, 1959). All chemicals used are from Sigma Company. The color of aerial and substrate mycelia grown on the different media used were recorded in addition to pigment production. Carbon utilization, nitrate reduction, melanin production, gelatin liquefaction and H2S production (Kuster & Williams, 1964) tests were used for physiological characterization.\n\nIn order to detect the production of extracellular hydrolases, different enzymatic agar plate assays were performed. The different assays are described below.\n\nThe starch agar medium was used to detect the amylase activity (Haritha et al., 2010). The assay medium inoculated with each isolates was incubated at 30°C for 72 hours. After incubation, the amylolytic activity was detected by flooding the agar plates with Gram’s iodine solution (2.0%). The change in color of clear zones around the growing colonies to dark blue was considered as positive.\n\nCellulase production was performed in agar plates supplemented with carboxymethyl cellulose (CMC) (0.5%) as the only carbon substrate, after incubation at 30°C for 72 hours. Three replicates were used for the each actinomycete isolates. The plates were then flooded with Congo red and NaCl. The yellow zones around colonies in respect to the red background indicated positive cellulose activity (Rathnan & Ambili, 2011).\n\nThe relative activity of protease production was detected for actinomycete isolates on milk agar plate, containing basal salt of starch casein amended with 20% of skimmed milk, following the method of Jani et al., (2012). The actinomycetes were grown in the middle of the milk agar plate and incubated for 5–6 days and at an interval of 24 hours. Zones of casein hydrolysis (clear zones) indicated positive results.\n\nTyrosinase activity was assessed in medium containing L-tyrosine (Sambasiva Rao et al., 2012). Plates containing L-tyrosine were inoculated with each tested isolate separately and then incubated at 37°C for 72 hours. The appearance of black or brown color around the margin of colonies and diffused to the medium indicated tyrosinase activity.\n\nTo observe lipase production, the actinomycetes were grown on modified medium of Vishnupriya et al., (2010) in which tween-20 (0.5%) was used instead of olive oil. Agar plates were inoculated and incubated at 30°C for 72 hours. The clearance zone around colonies was considered a positive evidence of tween-20 hydrolysis.\n\nAll isolates obtained were screened for catalase activity after 3 to 4 days of subculture on newly fresh SCM following the method of Mahon et al., (2011) using the slide (drop) method. Positive reactions were evident by immediate effervescence (bubble formation).\n\nAcid and alkaline phosphatase activities were determined according to Ghorbani-Nasrabadi et al., (2013). Inoculated medium supplemented with CaHPO4 (5 g/l) were incubated at 37°C for 48 to 72 hours. Phosphatase active isolates were recorded based on the halo-zones produced around the colonies.\n\nEvaluation of enzymatic activity. The enzymatic activity (EA) of different tested substrates was examined. The diameter of growth was measured and the clear zone representing enzyme activity was calculated by using the formula:\n\nEA = Diameter of zone of tested substrate hydrolysis - Diameter of colony in cm.\n\nBased on the EA test, the organisms can be categorized into three groups: showing excellent activity (EA>2), good (EA<2) and poor (EA<1).\n\n\nResults and discussion\n\nA total of 35 actinobacteria isolates were obtained from the two different soils collected from Northern part of Al-Aqsa mosque in Jerusalem. From Site 1, only 17 isolates were recovered in which four genera, Actinomadura, Streptomyces, Elylrosporangium and Actinopolyspora, were represented (Table 1). Site 2 was represented by more diverse genera of 18 isolates, Streptosporangium, Actinomadura, Nocardiopsis, Nocardia, Elytrosporangium and Actinopolyspora (Table 1). Genus Actinomadura was represented with the highest frequency in both sites (52.95% and 33.33% for site 1 and site 2 respectively). Despite the fact that isolation methods reveal only a minor fraction of the real existing microbes (Groth et al., 1999) we could demonstrate a great diversity among actinobacteria in the studied sites. To our knowledge (Thaer et al., 2009), this is the first study reporting identification of actinomycetes from this terrestrial environment. Meanwhile, the genera obtained are the first to be recorded in Jerusalem or in the whole country.\n\nThe differences observed among the genera of actinobacteria identified in both sites studied, may be due to the different human activities, including construction in such area. These differences may also indicate that the methods proposed and employed for actinobacteria isolation may not the suitable for site 1, and that soil pretreatment should be sought to explore the other genera inhabiting the area. Machavariani and colleagues (2011) found that preliminary treatment of soil by chemical substances like adrenaline and heteroauxin exerts a positive influence on the germination of actinomycete spores and contributes to the natural product activity of the isolated strains. Moreover, they demonstrated that soil pretreatment with different chemical substances play a role for the most complete isolation of actinomycetes that inhabit certain soil.\n\nWhen screening enzyme activities, 32 of the 35 actinobacteria isolate showed a good amylolytic activity (Figure 1A) and the other three isolates (site 2), Streptosporangium sp.2, Nocardia sp.2 and Elylrosporangium sp.4, had a moderate activity (Figure 1B). However, all isolates from site 1 had a good amylase activity (Table 1). For other enzymes, although actinomycetes are well known as potent degraders of cellulose, lignin, chitin and other complex polysaccharides (El-Fiky et al., 2003; McCarthy & Broda, 1984; Prasad et al., 2012; Wilson, 1992) none of our isolates were able to produce cellulase enzyme. Our results seem to be in contrast with previous studies, and this may be explained by the unsuitable culture conditions for cellulase production such as optimal pH as reported by (Rathnan & Ambili, 2011). In their study, they showed that cellulase enzyme production by Streptomyces sp. using fruit waste as substrate was the highest at alkaline pH.\n\n(A and B) showing good to moderate amylase activity respectively; (C and D) showing moderate protease activity indicated by clear zones around colonies; (E) showing good tyrosinase activity indicated by deep brown pigments and (F) showing no activity for two different actinomycete isolates.\n\nProteases represent one of the most important groups of enzymes and have been shown to play a role in many industrial and medical fields (Prakash et al., 2013). They can be used in detergent, food pharmaceutical, leather, waste processing industries and silk industries. Proteolytic enzymes have already been used in various forms of therapy and their use in medicine is gaining interest. Therefore, searching for new actinomycete strains still is the focus of many studies (Prakash et al., 2013). Our results revealed that only three isolates, Streptosporanium sp.1, Streptosporangium sp.2 and Actinomadura sp.10 were able to produce protease enzyme (Figure 1C and D). These three isolates were obtained from site 2. However, the site 1 and the reset of isolates from site 2 completely failed to show any protease activity. This rather low estimate of active strains may indicate that the method used for preliminary screening is not accurate (Jani et al., 2012). In addition, the low activity observed may suggest that our culture conditions are not optimal for such test (Jani et al., 2012).\n\nThe search for novel tyrosinases is still in need due to their potential in industrial applications and medical purposes. Tyrosinases have been suggested as potential tools in treating melanoma and as potential antioxidants, antiviral agents and immunogens (Popa & Bahrim, 2011). Among the isolates, only few showed tyrosine activity but all of these exhibited strong activity (Table 1). Only one isolate out of 17 (5.9%) in site 1, Elylrosporangium sp.3, showed high production of tyrosinase and the rest failed to show any activity (Figure 1E and F). Meanwhile, four isolates out of 18 (22.2%) of site 2, Streptosporangium sp.2, Kitasatosporia, Nocardia sp.2, and Actinomadura sp.15 showed different tyrosinase activity. These isolates had a high potential for enzyme production except Nocardia sp.2 which showed moderate activity.\n\nFor lipase production, all isolates from site 1 failed to show any activity. However, three isolates (16.7%) out of 18 isolates of site 2, Nocardiopsis sp.1, Nocardiopsis sp.2 and Kitasatosporia sp., were able to produce lipase in a weak to moderate manner of activity (Table 1).\n\nCatalases are ubiquitous enzymes and have been isolated from a broad range of procaryotic and eukaryotic organisms (Zámocký et al., 2012). Actinobacteria are aerobic bacteria and would be expected to have catalase activity; however, our actinomycete isolates recovered from both studied sites showed different ranges of positive activity (Figure 2). The catalase activity was observed at pH 7 of the growing medium for all isolates. Out of the isolated actinomycetes, Kitasatosporia sp. was the most active catalase producer, followed by 42.9% of the isolates have moderate catalase activity (Table 1). However, 18 isolates (51.4%) including different genera showed weak activity. The low expressed activity may be due to shortage of manganese ions or iron concentration in the growing medium, since catalases rely on iron or manganese for their activity (Mishra & Imlay, 2012). Further studies are needed to identify the factors that weaken the scavenger process.\n\nA and B show the activity of isolates from site 1 and site 2 respectively. Evolved oxygen bubbles indicated a very strong activity and weak bubbles indicated a weak activity.\n\nSince phosphate always exists in unavailable form for plant growth, phosphatase activity is an important mechanism of solubilizing inorganic phosphate (Sharma et al., 2013). In our study, all strains listed in Table 1, grown at room temperature (approximately 27±2°C) on basal medium supplemented with CaHPO4 (5 g/l) were tested for phosphatase activity at pH 7.0. Among the isolates, only Streptosporangium sp.1 and Nocardiopsis sp.2 showed phosphatase activity. The failure to detect of phosphatase activity by the remaining isolates may be due to the medium composition (Fredrikson et al., 2002). In a recent study done by Ghorbani-Nasrabadi et al., (2013), it was shown that substitution of nitrogen source in the growth medium by organic or inorganic nitrogen sources resulted in a significant reduction of phosphatase activity.\n\nIn conclusion, the need for low cost enzymes that can be applied in diverse biotechnological industries could be satisfied with the discovery of novel enzymes and metabolites. Moreover, the application of genetic engineering techniques in enzyme manufacturing is dramatically sparking the exploitation of new enzymes and the development of new enzyme properties. Actinobacteria have been proved a reservoir of important enzymes and metabolites due to their versatile genetic repertory (Prakash et al., 2013). Identification of new actinobacterial isolates in unique ecological environments could yield molecules that could become future players in green technology. Therefore our study contributes to explore new ecological sites for actinobacterial identification. Actinobacterial isolates that inhabitant the northern part of Al-Aqsa Mosque, showed a diverse population in the two sites where the soil was collected. These genera have been identified in each site for the first time. However more suitable growth conditions should be tested to explore the metabolites and enzymatic activities of these organisms.", "appendix": "Author contributions\n\n\n\nMansour S., Abedelazeium A. and Deraz S. conceived the study. MS and AA designed the experiments. MS and DS carried out the research. AA and MS provided the expertise throughout the experiment. MS prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors would like to thank Prof. Louis Tisa for revising the manuscript. Also thanks go to the researchers A. Salh and A. Hamza for their help during the lab work.\n\n\nReferences\n\nAnn MH, Maria V: Micromonospora: An important microbe for biomedicine and potentially for biocontrol and biofuels. Soil Biology & Biochemistry. 2010; 42(4): 536–542. Publisher Full Text\n\nAnon Bergey’s Manual of Systematic Bacteriology, 4. ed.Williams, S.T., Sharpe, M.E. and Holt, J.G. Baltimore, MD USA: Williams & Wilkins. 1989; 4. Reference Source\n\nCrawford DL: Biodegradation of agricultural and urban wastes. In : Actinomycetes in biotechnology, M.Goodfelow (Ed.), Academic Press, London. 1988; 433–459. Publisher Full Text\n\nEl-Fiky Z, Mansour SR, El-Zawhary Y, et al.: DNA-Fingerprints and phylogenetic studies of some chitinolytic actinomycete isolates. J Biotechnology. 2003; 2(2): 131–140. Publisher Full Text\n\nFredrikson M, Andlid T, Haikara A, et al.: Phytate degradation by micro-organisms in synthetic media and pea flour. J Appl Microbiol. 2002; 93(2): 197–204. PubMed Abstract \| Publisher Full Text\n\nGhorbani-Nasrabadi R, Greiner R, Alikhani HA, et al.: Distribution of actinomycetes in different soil ecosystems and effect of media composition on extracellular phosphatase activity. J Soil Sci Plant Nutr. 2013; 13(1): 223–236. Publisher Full Text\n\nGroth I, Vettermanna R, Schuetze B, et al.: Actinomycetes in Karstic caves of northern Spain (Altamira and Tito Bustillo). J Microbiol Methods. 1999; 36(1–2): 115–122. PubMed Abstract \| Publisher Full Text\n\nHaritha R, Siva Kumar K, Jagan Mohan YSYV, et al.: Amylolytic and proteolytic actinobacteria isolated from marine sediments of Bay of Bengal. International J Microbiol Res. 2010; 1(2): 37–44. Reference Source\n\nJani SA, Chudasama CJ, Patel DB, et al.: Optimization of extracellular protease production from alkali thermo tolerant actinomycetes: Saccharomonospora viridis SJ-21. Bull Environ Pharmacol Life Sci. 2012; 1(6): 84–92. Reference Source\n\nKandasamy S, Muthusamy G, Thangaswamy S, et al.: Screening and identification of antibiotic producing actinomycetes and their antagonistic activity against common pathogens. World Research Journal of Antimicrobial Agents. 2012; 1(1): 07–10.\n\nKuster E, Williams ST: Production of Hydrogen Sulfide by Streptomycetes and Methods for its Detection. Appl Microbiol. 1964; 12(1): 46–52. PubMed Abstract \| Free Full Text\n\nMachavariani N, Kustova N, Galatenko O: Isolation of actinomycetes - producers of antibiotics - from soil by selective methods based on activation of spore germination. Chemical Petroleum Engneering. 2011; 46(11–12): 667–669. Publisher Full Text\n\nMahon CR, Lehman DC, Manuselis G: Textbook of diagnostic microbiology, 4th ed. W. B Saunders Co., Philadelphia, PA. 2011. Reference Source\n\nMansour SR: The occurrence and distribution of soil actinomycetes in Saint Catherine area, South Sinai, Egypt. Pak J Biological Sci. 2003; 6(7): 721–728. Publisher Full Text\n\nMason MG, Ball AS, Reeder BJ, et al.: Extracellular heme peroxidases in actinomycetes: a case of mistaken identity. Appl Environ Microbiol. 2001; 67(10): 4512–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcCarthy AJ, Broda P: Screening for lignin-degrading actinornycetes and characterization of their activity against [I 4C]Lignin-labelled Wheat Lignocellulose. J General Microbiol. 1984; 130: 2905–2913. Reference Source\n\nMishra S, Imlay J: Why do bacteria use so many enzymes to scavenge hydrogen peroxide? Arch Biochem Biophys. 2012; 525(2): 145–160. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrakash D, Nawani M, Prakash M, et al.: Actinomycetes: a repertory of green catalysts with a potential revenue resource. Biomed Res Int. 2013; 2013: 1–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrakash D, Nawani M, Prakash M, et al.: Actinomycetes: a repertory of green catalysts with a potential revenue resource. Biomed Res Int. 2013; 2013: 1–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPopa C, Bahrim G: Streptomyces tyrosinase: Production and practical applications. Innovative Romanian Food Biotechnology. 2011; 8: 1–7. Reference Source\n\nPridham TG, Anderson P, Foley C, et al.: A selection of media for maintenance and taxonomic study of Streptomyces. Antibiot Annu. 1956–57; 947–953. 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PubMed Abstract\n\nSharma SB, Sayyed RZ, Mrugesh HT, et al.: Phosphate solubilizing microbes: sustainable approach for managing phosphorus deficiency in agricultural soils. Springer Plus. 2013; 2(578): 2–14. Publisher Full Text\n\nSolecka J, Joanna Z, Magdaiena P, et al.: Biologically active secondary metabolities from actinomycetes. Cent Eur J Biol. 2012; 7(3): 373–390. Publisher Full Text\n\nThaer A, Bapi RK, Ellaiah P: Antibacterial activity of bacterial isolates of soil bacteria collected from Palestine. Current Trends Biotechnol Pharmacy. 2009; 3(2): 197–203. Reference Source\n\nVishnupriya B, Sundaramoorthi C, Kalaivani M, et al.: Production of lipase from Streptomyces griseus and evaluation of Bioparameters. Chem Tech. 2010; 2(3): 1380–1383. Reference Source\n\nWaksman SA: The Actinomycetes classification, identification and determination of genera and species, The Williams and Wilkins Co., Baltimore, 1961; 2. : 1–363.\n\nWilson DB: Biochemistry and genetics of actinomycete cellulases. Crit Rev Biotechnol. 1992; 12(1–2): 45–63. PubMed Abstract \| Publisher Full Text\n\nZámocký M, Gasselhuber B, Furtmüller PG, et al.: Molecular evolution of hydrogen peroxide degrading enzymes. Arch Biochem Biophys. 2012; 525(2): 131–144. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "7375", "date": "20 Jan 2015", "name": "Jean-Jacques Sanglier", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSure it is of interest to evaluate the actinomycete populations of unique sites. However, the data should be sufficient and the goal has to be clearly defined and of interest.The isolation procedure of the strains do not allow to cover all the actinomycetes population. One has to apply pre-treatment(s)1 of the samples, use more media such as Humic Acid Vitamins Agar2 and the agar-plates have to be incubated at least 3 weeks. With the procedure used one can isolate only the fast growing strains.The identification of the strains to the genus level cannot be achieved only with the methods used. It could be tentatively possible for some genera but not in any case for all. Actinomycetes taxonomy was previously based on morphology, which is inadequate in differentiating between various genera3,4. Present approaches to the classification of prokaryotes are based on the combined use of genotypic and phenotypic data, that is, on polyphasic taxonomy5,6. This approach is being driven increasingly by molecular biology, especially the impact of 16S rRNA gene sequence and DNA:DNA relatedness values on the delineation of taxa7.“The differences observed among the genera of actinobacteria identified in both sites studied, may be due to the different human activities, including construction in such area.” The authors should not forget that they isolated only a very small part of the actinomycetale populations, so the comparison is difficult. The only way would be to use culture-independent methods.In addition according to Rule 37a, bacteriologists adhering to this proposal must change the name Elytrosporangium to Streptomyces.The fact that the isolated strains showed various enzymatic activities is not surprising and such an observation can be made with all samples. However the description of the activities is well done, even if basic. There are many publications in this area. So the goal should be the detection of a rare enzymatic activity or the comparison of the enzymatic potential of the actinomycetes from this site to other ones or to characterize the enzymes somehow.", "responses": [] }, { "id": "7319", "date": "29 Jan 2015", "name": "Giordano Rampioni", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript describes the isolation and preliminary characterization of actinobacteria strains producing enzymes with potential biotechnological applications. However, the experimental design chosen by the authors, and the conclusions drawn from some results rise some concerns.The major weakness of this work is that the assays used to investigate the enzymatic potential of the isolated actinobacteria are not fully convincing. Especially for what concerns cellulase activity, none of the strains isolated by the authors was positive to this test. The authors discuss that this unexpected result could be due to unsuitable culture conditions, such as pH. This is also the case for protease and catalase activities, that were found just in few isolates, and for which negative results could be biased by the experimental method. Authors should demonstrate that each enzymatic assay works in their hands, by using bacterial strains previously characterized for their ability and inability to produce the tested enzymes as positive and negative controls, respectively. Moreover, other experimental settings (e.g., cultures with different pH) should be considered to verify enzymatic activities in the isolates. The authors state that enzymatic activity (EA) was evaluated as follow: “diameter of zone of tested substrate hydrolysis - diameter of colony in cm”. Based on this calculation, the EA was estimated as excellent (EA>2), good (EA<2), or poor (EA<1). Besides noticing that EA<2 is not correct (it should be 1<EA<2), this “EA” values are not mentioned anymore along the manuscript, where the detected activity is usually defined as “strong activity”, “moderate activity” or “low activity”. In addition, in Table 1 the enzymatic activity is represented with + or – symbols. Authors should choose one adjective/word to define a certain interval of activity and use always the same along the whole text. The measurement of a diameter (see above) can be acceptable as a semi-quantitative method allowing the comparison of a certain enzymatic activity among different strains. However, since catalase activity was detected by visualizing oxygen bubbles formation, it is not clear how the authors measured and compared this enzymatic activity. Taxonomic allocation of the isolated strains should be confirmed by 16S rRNA sequencing. Since the main aim of this article is to explore the enzymatic potential of newly isolated actinobacteria, the authors were expected to use protocols allowing maximal diversity of the isolated species. The authors discuss that in previous studies sample pre-treatment with specific agents has been successfully used to promote spore germination and to allow isolation of most actinobacteria inhabiting the soil, so it is not clear why they did not consider this possibility for the experimental plan. Authors state that “the differences observed among the genera of actinobacteria identified in both sites studied, may be due to the different human activities, including construction in such area. These differences may also indicate that the methods proposed and employed for actinobacteria isolation may not the suitable for site 1,...”. In order to support this piece of discussion, authors should provide more information about the characteristics of the two isolation sites (e.g., anthropic impact, type of soil, type of vegetation). Were these sites similar or different with respect to one or more features? How these similarities/differences could be correlated to the results?", "responses": [] } ]	1	https://f1000research.com/articles/4-11
https://f1000research.com/articles/3-267/v1	06 Nov 14	{ "type": "Method Article", "title": "Safety, efficacy and utility of methods of transferring adhesive and cohesive Escherichia coli cells to microplates to avoid aerosols", "authors": [ "Bryan Ericksen" ], "abstract": "The virtual colony count (VCC) microbiological assay has been utilized for over a decade to measure the antimicrobial activity of peptides such as defensins and LL-37 against biosafety level (BSL)-1 and BSL-2 bacteria including Escherichia coli, Staphylococcus aureus, Bacillus cereus, and Enterobacter aerogenes. In addition, a modified pipetting technique was presented in a 2011 study of defensin activity against the BSL-3 pathogen Bacillus anthracis. Both studies were published in the journal Antimicrobial Agents and Chemotherapy. Here we report that the method can also detect cross-contamination caused by aerosols utilizing the VCC method of data analysis by quantitative growth kinetics (QGK). The QGK threshold time, or Tt, equivalent to the cycle time Ct reported in 1996 by Heid et al., precisely identifies when wells were inoculated.", "keywords": [ "Aerosols", "Aerobiology", "Quantitative Growth Kinetics", "Environmental Monitoring" ], "content": "Introduction\n\nThe virtual colony count (VCC) microbiological assay has been utilized for over a decade to measure the antimicrobial activity of peptides such as defensins. The initial VCC publication (Ericksen et al., 2005) used two methods of transferring cells to microplates using a 20–200 microliter multichannel pipettor: 22.2 microliters added to 200 microliters of media in calibration experiments and 50 microliters added to 50 microliters of solutions in phosphate buffer. Further experimentation has demonstrated that only the former method safely and effectively transfers cells to the intended wells, and the latter method can result in cross-contamination.\n\nThe reason for this difference is that adding cells suspended in 50 microliters directly to a like volume caused unacceptable froth, bubbles and background turbidity that is incompatible with the VCC method of measuring growth kinetics by an increase in optical density using a 96-well plate in a plate reader. This problem, which affects optical density readings in turbidimetric assays, was initially solved by holding pipette tips just above the liquid but below the rims of the wells and adding cell suspensions as droplets. Accurately holding the multichannel pipettor within this narrow range seemed to require placing one’s eyes as close as possible to the 96-well plate, but further experiments using biosafety cabinets have proven that the method can be done by a well-trained operator looking through the glass. Assays conducted in 2012 and 2013 within a biosafety cabinet at the University of Maryland Baltimore (UMB) resulted in frequent cross-contamination of the 36 contamination control edge wells. Light microscopy revealed adhesive and cohesive clumps and biofilms formed by Escherichia coli ATCC 25922 and Staphycococcus aureus ATCC 29213. Changes in particle size distribution and adhesive properties due to clumping apparently resulted in increased aerosol formation, which made cross-contamination far more common than in the initial studies in 2003–2004 preceding the 2005 publication of VCC. Using this procedure for hazardous microorganisms outside a biosafety cabinet would pose a safety risk.\n\nThe VCC plate configuration as initially published in 2005 used the 36 wells around the edge of the 96-well plate (rows A and H and columns 1 and 12) as contamination control wells. Turbidity in these wells could have been the result of either environmental contamination or cross-contamination, but sampling wells over the course of many experiments revealed colony morphologies that were almost invariably consistent with the bacterial strain studied that day. Six alternating VCC experiments using Escherichia coli ATCC 25922 and Staphylococcus aureus confirmed this conclusion by producing colonies only consistent with the strain studied that day, not the strain studied in the previous experiment or an environmental isolate with a colony morphology matching neither strain.\n\nTwo hypotheses regarding the origin of cross-contamination were pursued: cells emanating from the pipette tips as they were passed directly over the contamination control wells or cells ejected up out of the wells as aerosols when the cell suspension was expelled. To distinguish between these possibilities, 13 experiments were conducted not with a single ring of 36 contamination control wells around the edge, but with an additional ring (columns 2 and 11 and rows B and G), totaling 64 uninoculated wells. In these experiments, quadruplicate 8-point 10-fold calibration dilutions were made by adding 22.2 microliters beneath 200 microliters of media, pipetting up and down 15 times, expelling tips, transferring 22.2 microliters to the next column of four wells, etc. None of the 832 uninoculated wells turned turbid after overnight incubation at 37 degrees shaking in a Tecan Infinite M1000 plate reader, indicating a lack of cross-contamination or environmental contamination that is viable in rich media originating from the laboratory, reagents, operator or reader. Next, several VCC experiments were conducted using eight cross-contamination control wells in column 12 with controls lacking antimicrobial agents in column 11 as described in the initial 2005 paper, during which all 24 cross-contamination control wells in column 12 turned turbid in all three experiments. Four changes were made to the procedure in an attempt to remove possible sources of contamination that may have caused cells to become more adhesive and cohesive, which in turn would have caused cross-contamination to become far more likely: 1. using a small HEPA-filtered air purifier, 2. replacing in-house deionized Milli-Q water with purchased molecular biology grade water, 3. replacing 2XMHB prepared and autoclaved in-house using reusable jars with Teknova 2X cation-adjusted MHB, and 4. filter-sterilizing phosphate buffers made near the portable air purifier, rather than autoclaving in reusable jars. After those changes, 25 mL TSB cultures of Escherichia coli ATCC 25922 grown simultaneously as a biosensor no longer produced macroscopic clumps with diameters on the scale of millimeters. However, cross-contamination in VCC experiments persisted. In several of these experiments, a separate 96-well plate containing media only was interposed between the reagent reservoir containing the cell suspension and the experimental 96-well plate, and in no case did any well in these additional plates turn turbid. Had cells been transiently adhering to the outsides of the tips or trailing from the liquid held by capillary action at the openings of the tips, many or all of the 96 wells of the cross-contamination plates would have turned turbid, since all cross-contamination wells in column 12 on the right edges of experimental plates turned turbid. Therefore, contamination caused by passing the tips over these wells without expelling was ruled out. The next simplest explanation is that, while the plunger of the multichannel pipettor was depressed to deliver cells as droplets below the rims but above the liquid in the wells, the tips expelled viable aerosols that travelled in an upward trajectory and escaped the intended wells in such great numbers that the cross-contamination of adjacent wells was probable to the point of inevitability.\n\n\nResults\n\nIn Experiment 1, all eight wells in column 12 turned turbid and produced growth curves with the same growth rate and doubling times as the other growth curves on the same microplate (Figure 1). Colony morphologies of samples from these wells also matched E. coli ATCC 25922. A comparison of threshold times indicated almost the same difference between input and output controls in columns 11 and 12 (Table 1). There was a roughly 70-minute difference in input and output threshold times in the input and output control wells in Experiment 1, which agreed closely with another roughly 70-minute difference in the threshold times of the adjacent wells. Contamination caused by environmental factors would have been expected to produce widely varying threshold times, if not visible differences in the appearance of the turbid wells. Therefore, the 70-minute difference indicated that the cross-contamination occurred at the same time that cells were transferred.\n\nUncorrected growth kinetics of columns 11 (panel A) and 12 (panel B) of the 96-well plate in Experiment 1. In these two columns (n=16), the threshold ΔOD650 value of 0.02 corresponded to a mean ± standard deviation uncorrected OD650 of 0.0989±0.0043, which corresponds to a %RSD of 4.4. The line marked “0.1” is approximately at the position of the threshold ΔOD650 of 0.02.\n\nA11-D11 are the “input” control wells and E11-H11 are “output” control wells. Cells were added to those two wells two hours apart, resulting in a 73.3 minute difference in Tt values. Cross-contaminated wells gave a corresponding Tt difference of 71.2 minutes, indicating that A12-D12 were inoculated as cells were being expelled over A11-D11, and E12-H12 were inoculated as cells were being expelled over E11-H11.\n\nIn Experiment 2, the threshold times again reflected a roughly 70-minute difference between input and output controls. (Figure 2 and Table 2) However, this difference was not reflected in threshold times of the cells growing in column 12, indicating that the contamination of those wells was the result of a second contamination event unrelated to the timing of the transfer of cells into the wells in column 10. The only reasonable explanation of this agreement in threshold time differences between columns 10 and 11 and the far larger Tt values resulting from column 12 is that cross-contamination occurred while cells were expelled, and the aerosols thus formed travelled to the adjacent wells but not the wells in column 12 or the intervening 96 contamination control wells in the contamination control plate, none of which turned turbid after overnight incubation at 37 degrees. These results indicate that 96-well plates and threshold times are useful for detecting contamination, and that cross-contamination occurs in experiments where cells are added as droplets from above.\n\nUncorrected growth kinetics of columns 10 (panel A), 11 (panel B) and 12 (panel C) of the 96-well plate in Experiment 2. In these three columns excluding well A12 (n=23), the threshold ΔOD650 value of 0.02 corresponded to a mean ± standard deviation uncorrected OD650 of 0.0988±0.0053, which corresponds to a %RSD of 5.4. The biphasic curve in well A12 was unique among the 96 wells analyzed in this assay, and is caused by an initial phase of optical density increase caused by condensation on the lid followed by a second phase caused by increased turbidity due to cell growth within the well.\n\nA10-D10 are the “input” control wells and E10-H10 are “output” control wells. Cells were added to those two wells two hours apart, resulting in a 68.2 minute difference in Tt values. Cross-contaminated wells gave a corresponding Tt difference of 42.4 minutes. The difference between these two values, 25.8 minutes, could be accounted for by the growth of additional cells added in a second contamination event reflected by wells B12-H12 Tt values that also caused media in the reservoir to turn turbid when collected and incubated overnight. Thus, Tt values detect cross-contamination in adjacent wells and can distinguish between separate contamination events.\n\n\nDiscussion\n\nThe method of enumeration of cells in a VCC assay is confounded if the cells form clumps, because that clumping and biofilm formation affects optical density readings. Other experiments revealed macroscopic clumps and biofilms visible to the unaided eye. In addition, microscopic clumps were revealed by light microscopy. Cohesion, adhesion, clumps and biofilms affect not only threshold times but also the particle size distribution of the cell suspension and the degree of adhesion as the cells are expelled through the pipette tips. Therefore, both clumps suspended in solution formed by cells adhering to each other but not surfaces and adhesive cells could affect the physical properties of the liquid as it is transformed to an emulsion that generates aerosols. Cross-contamination was far more common in the experiments I conducted in 2012–2013 compared to experiments I conducted in 2003–2004 in an adjacent room, suggesting that some change in environmental factors between those times or locations caused greater cell clumping and adhesion, which in turn greatly increased the probability that a cross-contamination control well would become turbid. It is postulated that one or more clumping environmental factor (CEF) is responsible for the change in cross-contamination and a 23-fold fluctuation in virtual lethal dose values reported by the HNP1 positive controls of the assay throughout E. coli ATCC 25922 experiments in 2013.\n\nIn 2011, a modified VCC procedure (Welkos et al., 2011) was published for use with the BSL-3 pathogen Bacillus anthracis, based on the procedure originally developed at UCLA in the laboratory of Robert I. Lehrer. The 50 microliter cell transfer step mentioned in the 2005 publication and used at the University of Maryland was replaced with the addition of cells suspended in a smaller volume of liquid, 10 microliters, added to 90 microliters of buffer. This procedure, similar to the calibration experiments detailed in the original VCC publication, did not generate unacceptable turbidity when cell suspensions were added with the tips placed at the bases of the wells beneath the buffer when it was tested in 2013 in the Institute of Human Virology building at IHV. Adding cell suspensions under liquid apparently greatly reduces the probability of aerosol formation, which is of concern not only for safety reasons, but also because the aerosol cloud within the well can alter experimental results by generating cells that adhere to the sides of the well during the exposure to the antimicrobial agent, then drop down to inoculate the outgrowth media after the antimicrobial peptides have been neutralized by broth during 12 hours of vigorous shaking within the plate reader. VCC users are cautioned to use the 2011 procedure, not the 2005 procedure, to add experimental cell suspensions. Following the 2005 procedure to add Staphylococcus aureus cell suspensions in droplets above the liquid in the wells rather than injecting the cell suspension beneath the liquid in the wells could expose the eyes to aerosols containing a biosafety level 2 pathogen that could cause blepharitis, corneal stromal microabscess, stromal edema, uveitis, ocular necrotizing fascitis, and blindness. (Boto-de-Los-Bueis et al., 2014; Shield et al., 2013) Biosafety level 2 precautions such as those recommended by the Centers for Disease Control in Biosafety in Microbiological and Biomedical Laboratories, 5th Edition (Miller et al., 2012) should be taken for any study of Stapylococcus aureus, including the safer 2011 VCC procedure.\n\nThese results highlight an advantage of using the VCC data analysis procedure of enumerating cells (Brewster, 2003), termed quantitative growth kinetics (QGK) by analogy to quantitative polymerase chain reaction (QPCR). (Heid et al., 1996) QGK and QPCR use a mathematically identical procedure for quantifying the initial number of cells or amplicons that were present at the start of the assay. The QGK threshold time Tt is equivalent to the PCR cycle time Ct. Calculating Tt values in the two experiments reported here unequivocally identified the time of the contamination event, gave quantitative batch culture growth kinetic data that suggested that the contamination was cross-contamination, and distinguished between two cross-contamination events. These features of QGK would greatly improve the quality of environmental monitoring data when used to detect contamination by aerosols or ambient viable microorganisms compared to turbidity measurements in the absence of a plate reader or observing the appearance of colonies on agar plates, neither of which provides kinetic data.\n\nFinally, it should be emphasized that the simple improvement of adding cells beneath liquid simultaneously achieves two useful changes at once, reducing the probability that cells inoculate wells other than the ones intended while simultaneously also limiting the probability that cells escape the 96-well plate entirely. Although the reason why the addition of 50 microliters of cells beneath 50 microliters of liquid was unacceptable in VCC experiments stemmed from the turbidimetric nature of the assay, this method of preventing cross-contamination is far from trivial or confined to VCC assays. It teaches a technical lesson limited not just to environments where airborne CEFs are present, but broadly applicable to all experiments where microbes are transferred using pipette tips, thereby potentially improving the usefulness of a wide range of laboratory procedures that might otherwise generate aerosols. Any change in a procedure that improves its safety and efficacy also improves its utility ad oculos.\n\n\nMaterials and methods\n\nVCC assays were conducted as described (Ericksen et al., 2005) and modified (Zhao et al., 2013). Twice-concentrated cation-adjusted Meuller Hinton Broth was purchased from Teknova, Inc. Phosphate buffers were made using Sigma monobasic and dibasic sodium phosphate dissolved in molecular biology grade water or equivalent purchased from multiple sources. Rainin GreenPak LTS 200 microliter filter tips were used with an eight-channel 20–200 microliter pipettor. Costar 3595 96-well plates were analyzed in a Tecan Infinite M1000 plate reader at 37°C.\n\nTwo experiments were conducted using Escherichia coli ATCC 25922. In Experiment 1, four each of “input” and “output” controls were placed in column 11 of the 96-well plate, with eight cross-contamination control wells in column 12. Wells A11-D11 contained controls in wells added at the time the cells were exposed to antimicrobial agents, termed the “output” controls, and equivalent to the controls mentioned in the initial 2005 publication. In addition, wells E11-H11 contained identical controls that had been stored on ice during the two-hour exposure to antimicrobial agents in phosphate buffer, termed the “input” controls because their Tt values represent the concentration of cells that were present when they were put into the assay at the start of the two-hour incubation. Since the antimicrobial assay is beyond the scope of this report, which focuses only on aerosol cross-contamination, columns 1–10 and the antimicrobial agents therein will not be discussed here.\n\nNext, in Experiment 2, the controls lacking antimicrobial agents were moved from column 11 to column 10, and columns 11 and 12 contained 16 uninoculated contamination control wells. Wells A10-D10 contained controls in wells added at the time the cells were exposed to antimicrobial agents, termed the “output” controls, and equivalent to the controls mentioned in the initial 2005 publication. In addition, wells E10-H10 contained identical controls that had been stored on ice during the two-hour exposure to antimicrobial agents in phosphate buffer, termed the “input” controls. These controls are designed such that comparing the difference in threshold times between the input and output controls, relating that difference to the calibration curve elsewhere on the same 96-well plate, and assuming that adhesion or cohesion and lag phases in exponential growth were the same for all cells, the growth of the cells during the two hour incubation on the plate could be quantified. Cells grow during the two hour incubation step, so enumerating the change in cell concentration during that step would allow the calculation of the difference in virtual survival values that would correspond to bacteriostatic activity.\n\n\nData availability\n\nF1000Research: Dataset 1. Growth kinetics optical density readings for Experiments 1 and 2, 10.5256/f1000research.5659.d38055 (Ericksen, 2014).", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nI thank Peprotech, Inc. for funding.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nI thank Robert I. Lehrer for helpful comments.\n\n\nReferences\n\nBoto-de-Los-Bueis A, Del Hierro Zarzuelo A, García Perea A, et al.: Staphylococcus aureus Blepharitis Associated with Multiple Corneal Stromal Microabscess, Stromal Edema, and Uveitis. Ocul Immunol Inflamm. 2014. PubMed Abstract \| Publisher Full Text\n\nBrewster JD: A simple micro-growth assay for enumerating bacteria. J Microbiol Methods. 2003; 53(1): 77–86. PubMed Abstract \| Publisher Full Text\n\nEricksen B: Dataset 1 in: Safety, efficacy and utility of methods of transferring adhesive and cohesive Escherichia coli cells to microplates to avoid aerosols. F1000Research. 2014. Data Source\n\nEricksen B, Wu Z, Lu W, et al.: Antibacterial activity and specificity of the six human {alpha}-defensins. Antimicrob Agents Chemother. 2005; 49(1): 269–275. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeid CA, Stevens J, Livak KJ, et al.: Real time quantitative PCR. Genome Res. 1996; 6(10): 986–994. PubMed Abstract \| Publisher Full Text\n\nMiller JM, Astles R, Baszler T, et al.: Guidelines for safe work practices in human and animal medical diagnostic laboratories. Recommendations of a CDC-convened, Biosafety Blue Ribbon Panel. MMWR Surveill Summ. 2012; 61(Suppl): 1–102. Erratum 61(14): 214. PubMed Abstract\n\nShield DR, Servat J, Paul S, et al.: Periocular necrotizing fasciitis causing blindness. JAMA Ophthalmol. 2013; 131(9): 1225–1227. PubMed Abstract \| Publisher Full Text\n\nWelkos S, Cote CK, Hahn U, et al.: Humanized theta-defensins (retrocyclins) enhance macrophage performance and protect mice from experimental anthrax infections. Antimicrob Agents Chemother. 2011; 55(9): 4238–4250. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhao L, Tolbert WD, Ericksen B, et al.: Single, double and quadruple alanine substitutions at oligomeric interfaces identify hydrophobicity as the key determinant of human neutrophil alpha defensin HNP1 function. PLoS One. 2013; 8(11): e78937. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "6659", "date": "26 Nov 2014", "name": "Dipshikha Chakravortty", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript titled \"Safety, efficacy and utility of methods of transferring adhesive and cohesive Escherichia coli cells to microplates to avoid aerosols\" by Bryan Ericksen reports a safer method for transferring certain strains of E.coli. Though this report may generate some interest the E.coli community, this paper will lack the diverse readership. Being a very focused Method paper, it is good to seek journals which reports methods. As the load of microorganisms are very different in different countries, to adapt this technique for any lab will not be possible. It is good to have a lab standard using this technique and it will be restricted to that lab. Finally this entire concept is very qualitative.", "responses": [ { "c_id": "1096", "date": "26 Nov 2014", "name": "Bryan Ericksen", "role": "Author Response", "response": "Dear Dr. Chakravortty,Thank you for taking the time to read the paper and submit your review. I am not sure what you mean by \"load of microorganisms\" or why you say these methods could not be adapted for other laboratories, but your comment regarding QGK and VCC lab-to-lab variability is well-taken, which would be an excellent topic for further study if the identical experiment were to be repeated in many laboratory environments around the world. That is one reason why encouraging safe pipetting techniques is so important. Although I only presented E. coli data using a biosafety level 1 (BSL-1) strain in the figures and tables, the consequences of aerosol transmssion are perhaps greater for more dangerous microorganisms such as the BSL-2 pathogen Staphylococcus aureus, which was mentioned in the text. QGK generally, and the VCC method specifically, could potentially apply to a wide variety of other bacteria, as has already been demonstrated. The references mentioned in this work include the results of experiments performed using Enterobacter aerogenes, Bacillus cereus, and Bacillus anthracis.However, your comment regarding the qualitative nature of threshold times is rather baffling. It is precisely the quantitative, rather than qualitative, nature of QGK that permits its use to determine precisely when a turbid well was inoculated and thereby confirm aerosol transmission at the time cells were expelled as the mechanism of cross-contamination. I would encourage a more careful reading of the paper and tables 1 and 2, which present quantitative, rather than qualitative, results.Finally, I should explain why this methodological paper is so narrowly focused, as you noticed. Its genesis was an erratum to the original VCC paper (cited here as Ericksen 2005). Here is the erratum text verbatim, which contains a critical methodological detail:\"The virtual colony count (VCC) microbiological assay has been utilized for over a decade to measure the antimicrobial activity of peptides such as defensins. The initial VCC publication used two methods of transferring cells to microplates using a 20-200 microliter multichannel pipettor: 22.2 microliters added to 200 microliters of media in calibration experiments and 50 microliters added to 50 microliters of solutions in phosphate buffer. Further experimentation has demonstrated that only the former method safely and effectively transfers cells to the intended wells, and the latter method can result in cross-contamination. The reason for this difference is that adding cells suspended in 50 microliters directly to a like volume causes unacceptable froth, bubbles and background turbidity that is incompatible with the VCC method of measuring growth kinetics by an increase in optical density using a 96-well plate in a plate reader. This problem, unique to turbidimetric assays, was initially solved by holding pipette tips just above the liquid but below the rims of the wells and adding cell suspensions as droplets. Assays conducted in 2012 and 2013 within a biosafety cabinet at the University of Maryland Baltimore (UMB) resulted in frequent cross-contamination of the 36 contamination control edge wells. Light microscopy revealed adhesive and cohesive clumps and biofilms formed by Escherichia coli ATCC 25922 and Staphycococcus aureus ATCC 29213. Changes in particle size distribution and adhesive properties due to clumping apparently resulted in increased aerosol formation, which made cross-contamination far more common than in the initial studies in 2003-2004 preceding the 2005 publication of VCC. Using this procedure for hazardous microorganisms outside a biosafety cabinet would pose a safety risk. In 2011, a modified VCC procedure was published for use with the BSL-3 pathogen Bacillus anthracis, based on the procedure originally developed at UCLA in the laboratory of Robert I. Lehrer. The 50 microliter cell transfer step was replaced with the addition of cells suspended in a smaller volume of liquid, 10 microliters, added to 90 microliters of buffer. This procedure, similar to the calibration experiments detailed in the original VCC publication, did not generate unacceptable turbidity when cell suspensions were added with the tips placed at the bases of the wells beneath the buffer. Adding cell suspensions under liquid greatly reduces the probability of cross-contamination., which is of concern not only for safety reasons, but also because the aerosol cloud within the well can alter experimental results by generating cells that adhere to the sides of the well during the exposure to the antimicrobial agent, then drop down to inoculate the outgrowth media after the antimicrobial peptides have been neutralized by broth during 12 hours of vigorous shaking within the plate reader. VCC users are cautioned to use the 2011 procedure, not the 2005 procedure, to add experimental cell suspensions.\"All four authors of the 2005 paper agreed to the above erratum text earlier this year. In rejecting the erratum, the journal suggested conducting further experiments and submitting a separate paper, which I can no longer do, since I am no longer employed by the lab. However, in previously conducted experiments I did not observe unacceptable froth and turbidity when I added a small volume of E. coli cells beneath liquid following the 2011 VCC method, and I had already proven that the 2005 VCC method resulted in cross-contamination due to aerosol formation based on experiments I conducted in 2013. Therefore, I wrote a draft of this paper and submitted it to Antimicrobial Agents and Chemotherapy, on the principle that errors reported in a given journal should be corrected in the same journal. Predictably, the paper was also rejected as too narrow. I have served as a reviewer for the journal, and I would have agreed had I not known about the rejection of the erratum. I appreciate the opportunity given to me by F1000Research to correct the literature in a separate peer-reviewed forum, which will have to suffice in place of an erratum to the original paper, and as a fortunate by-product of this process I have also had the opportunity to present a novel way of quantifying cross-contamination. I believe aerosol transmission could explain some nosocomial infections and affect the experimental results of other assays where liquid is transferred, so I hope this modest paper will benefit the fields of assay development, bacteriology and aerobiology in some small way.Sincerely,Bryan Ericksen, Ph.D." } ] }, { "id": "6656", "date": "27 Nov 2014", "name": "Lynn Silver", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe Virtual Colony Count was devised in the author’s laboratory and has been in use for over ten years. It seems that this would be a very useful method for many applications as it can obviate the need for actual colony counting while still providing information about viability [although not “cfu” per se]. It would be useful to better introduce the system with a short explanation of the method itself so that new readers can see its potential benefits.This paper is a careful demonstration of the source of contamination – the production of aerosols – which have occurred when, as in the original method, 50 microliters of cell suspension is delivered above the surface of 50 microliters of test compound in medium (although this was not problematic during the initial work). Not surprisingly, contamination, and by deduction, aerosols were greatly reduced when a smaller volume, 22.2 microliters, was pipetted below the surface of 200 microliters of medium. Is it known if the latter procedure leads to better initial mixing than the former, as might be expected? A set of 13 preliminary experiments was carried out to generate hypotheses about the origin of contamination seen in medium control wells, as outlined in the introduction. It might be useful to show schematics of the plate formats used since following the details in the introduction is a little confusing. The same is true for the two final experiments that were performed to test the ultimate hypothesis. Also the terms input and output controls could be defined better. The difference between the 2012-2013 and 2003-2004 results is ascribed to “one or more clumping environmental factors (CEF)”. First, why abbreviate the name to CEF as it is only used twice in the paper? Second, is it possible that differences could be due to changes in the labware used, such as pipette tips, pipettors, microtiter plates? Even when purchased from the same vendor and ostensibly the same over time, it is possible that slight manufacturing changes could affect the results. For example, the 2013 set up might lead to less controllable expulsion of liquid. The experiments are well planned and the work is carefully done.", "responses": [] } ]	1	https://f1000research.com/articles/3-267
https://f1000research.com/articles/4-10/v1	12 Jan 15	{ "type": "Research Article", "title": "Possible significance of spatial heterogeneities of local visual features for face perception", "authors": [ "Vitaly V. Babenko", "Daria S. Alekseeva", "Denis V. Yavna", "Vitaly V. Babenko", "Daria S. Alekseeva" ], "abstract": "Second-order visual filters are the mechanisms which preattentively combine the rectified outputs of first-order filters (the linear striate neurons). This allows them to select the image areas which are characterized by spatial heterogeneity of the local visual features. The aim of our research is to determine whether information from these areas may be sufficient to detect unfamiliar faces and to distinguish their gender. In our experiments we used digital photos of real living things or artificial objects and faces. All these images were adjusted to an average luminance, contrast and size (7 angle degree) and were processed to extract the areas which differ the most in contrast, orientation, and spatial frequency in each of the six spatial frequencies (0.5, 1, 2, 4, 8, and 16 cpd). The other image parts were adjusted to the background. The obtained pictures were presented in a random sequence. The observer had to say what he/she saw after each presentation. When a face was presented the observer’s answer could be assigned to one of the categories: ‘it is not clear’, ‘head’, ‘human face’, ‘male / female’. We found that the information contained in the image areas with a spatial heterogeneity of the local features is sufficient not only for detecting a face, but also for distinguishing its gender. The best results were obtained at a carrier frequency of 2 cpd. The results were a little bit worse at 0.5 and 1 cpd. However, the information extracted from the high-frequency half of the spectrum was significantly less useful. The obtained results allow us to suggest that the information transmitted by the second-order visual filters may be used for pattern recognition.", "keywords": [ "visual filters", "spatial heterogeneity", "pattern recognition" ], "content": "Introduction\n\nThe issue of visual image formation has a long history. Until recently, there hasn’t been proposed a theory that could explain everything on that matter. In visual neuroscience 3 points of view on image formation have prevailed. According to the first one, an image is a holistic description in which the most typical object of its class is taken as a standard. This is called ‘a template theory’. The second point of view also came from a holistic image description, but takes an average description of an object of its class as standard. This is called ‘a prototype theory’. The third theory assumes that every image can be described using the summation of its features. This is called ‘a feature theory’. Until now it’s remained unknown how could the invariance of holistic descriptions be provided and what could be presented as separation features.\n\nNo matter what point of view is closer to the truth, it is now stated for sure that the initial visual processing is a parallel local description of an input, which results in breaking a scene into a quantity of fragments, which are known as primitives. These are the gradients of luminance of various localization, orientation and spacial frequency. The operation begins in retina and ends in the visual cortex.\n\nBut this is only the start of visual processing. Image formation inevitably includes grouping of primitives, attributed to a single object. At first, the theory of the integration of features, according to which the mechanism of bounding is selective attention1, was popular. Lately though, the number of tasks had been described to solve which spacial grouping implements preattentively. It is, for example, perceiving of second order movement2 and texture separation3. These operations can be done by the so-called ‘second-order mechanisms’ that preattentively (following a certain rule) bind outputs of first-order mechanisms (the linear striate neurons)3–5. The following studies proved an existence of such mechanisms and determined its properties6–9. Considering the second order movement to be a laboratory phenomenon, the ability to quickly divide textures is very important in everyday life.\n\nIs the role of the second order mechanisms limited by the task of texture separation? Considering that these mechanisms can distinguish special modulations of local features, the attempts were made to establish the considerable role of this information in perceiving complex scenes and objects. Analysis of the natural pictures showed that notably the first and the second order features spatially overlap10,11. As a result the second order features were determined to delete the ambiguities from interpretation of the change of luminance (the first order features)12,13.\n\nMeanwhile, it’s quite rational to assume that these modulations could contain important information concerning an object’s forms and their details. Considering this, the goal of our study – to determine whether information concerning spacial heterogeneity – could be useful in identifying all the images and faces among the ‘not faces’ in particular.\n\nInitiating the task ahead, we cannot ignore the fact that the early visual processing is operated by the system of parallel paths that are set to different spacial frequencies14–17. It is known that, when it comes to tasks of identifying faces, these frequencies are not the same. There’s also the probability that the results of the processing by particular spacial channel are united in certain combinations.\n\nPreparing the test images, we followed the assumptions about the organization of the second order mechanisms, which are displayed in the ‘filter-rectify-filter’ model18. According to it, the outputs from adjacent linear filters (the first-order filters) with the same frequency and orientation set are united by the certain algorithm in the second-order filters. In other words, in case of the second-order mechanisms, those filters that differ only in localization in field of view unite. Such filters with different resolutions pass those regions of image that differ in heterogeneity to contrast, orientation and spacial frequency. We followed the assumption that these regions, due to their heterogeneity, contain the important information and could be viewed as the ‘regions of interest’.\n\n\nMethods\n\nThe stimuli were displayed on a 17” LG Flatron 775FT monitor hosted by a PC (amd64-compatible) with an NVIDIA GeForce 7300 SE graphical subsystem running Debian GNU/Linux 7.2 (wheezy). The screen resolution was 1152 × 864 pixels with a refresh rate of 75 Hz. The monitor luminance was calibrated by a digital photometer (manufactured by ‘TKA’, St. Peterburg, Russia) using 256 gray levels.\n\nThe digital photographs of real objects and faces were used as initial images. All images were previously adjusted in size (7 angle deg.). The average luminance of the stimulus equaled the luminance of the background and was 19 kd/m2. Initial images were processed in such a way that the areas which were different from the surroundings in contrast, orientation and spatial frequency in 6 frequency ranges corresponding to the frequency tuning of human visual pathways were extracted19. The object size was such that its maximum length along any axis corresponded to 0.5 period of the SOF (the window diameter) which was tuned to the lowest carrier frequency (3 cpi).\n\nThe sequence of the computations for the preparation of the test images reproduced the operation sequence in the basic model ‘filter-rectify-filter’:\n\nThe initial image linear filtration (by FOFs).\n\nThe FOF’ core is a two-dimensional Gabor function20,21. FOF bandpass is 2 octaves. 6 peak spatial frequency with an increment of 1 octave (from 4 to 128 cpi) and 6 orientations with an increment of 30 deg. (from 0 to 150 deg.).\n\nRectification.\n\nThe rectification was realized by square-rooting of the sum of squares of the FOFs‘ outputs forming the quadrature pair.\n\nThe linear filtering of the 36 obtained images (6 spatial frequencies × 6 orientations) by the SOFs.\n\nThe SOF’ core is a two-dimensional Gabor function with 1 period which is 8 times longer than 1 period of the combined FOFs9,13. The orientation tunings of the FOF and SOF were the same22–24.\n\nOrientation integration.\n\n6 values corresponding to 6 FOF’ orientations were obtained for each pixel. Then the maximum of these values was attached to each pixel.\n\nFinding the local peaks at the SOFs‘ outputs.\n\nThe local maximums were found in each of the six matrices of the SOFs‘ outputs (6 spatial frequencies of the carrier).\n\nWindows allocation.\n\nEach maximum in each ‘frequency slice’ became the window center through which the information from the FOFs was allowed to pass. The window’s diameter was 0.5 of the period of the SOFs forming this frequency slice.\n\nFilling of the windows.\n\nEach window was filled with the image, obtained by FOFs at corresponding frequency. The pixels‘ luminance was decreased by Gaussian from the window center to the periphery. The image was filled with the background outside the window. In the case of overlapping of the windows the pixels got the major luminance value.\n\nThe subjects were seated at the distance of 1.15 m from the monitor that was randomly showing the previously made stimulus. Looking at the queue image, the observer needed to tell what he saw. The time of showing wasn’t limited. The images based on photo of man’s and women’s face (unfamiliar) were shown in the queue of the ‘not-faces’ images. The subject’s responses to the above-mentioned images could’ve been categorized in one of three existing categories (‘head’, ‘human’s face’, ‘man or women’), or said that it hadn’t been noted at all in case of a wrong or missing answer. Questions that could lead the observer to the right answer were not asked.\n\nA total number of 70 students (9 men and 61 women) aged between 17 and 21 took part in this experiment. All the participants had normal or corrected to normal vision and no history of neurological or psychiatric disorders had been reported. The participants did not take any medicines just before or during the study tests. All the participants of the research were informed about the purpose and the procedures of the experiment; they all signed a consent form that outlined the risks and benefits of participating in the study and indicated that they believed in the safety of the investigation. The study was realized in accordance with the ethical standards consistent with The Code of Ethics of the World Medical Association (Declaration of Helsinki) and approved by the local ethics committee.\n\n\nResults\n\nThe information was allowed to pass only through one ‘window’, centered relative to the face (Figure 1A,C) when the initial (real) images were processed by the SOFs tuned to the lowest frequency of the carrier (0.5 cpd) (we denote these filters as F1). The result of the processing is shown in Figure 1B,D. Looking at the presented images the observers determined the gender in 87.9% and gave a more general response “a face” only in 11.4%.\n\nA, C – the initial images. The circles are the windows through which the filtered image is allowed to pass. B, D – the test (processed) images. There are only ‘the regions of interest’ at the frequency of filtering.\n\nIf the initial images were processed by the SOFs tuned to a higher frequency of the carrier (1 cpd) (F2) the information from only a part of a face could be transmitted through one window (Figure 2A,C). The information was transmitted through 2 windows because there were 2 local maximums at the F2 outputs. The result of the processing may be seen in Figure 2B,D. We should mention that the observers’ results were a little worse than the previous ones. Now the gender was defined in 75.7% and the response ‘a face’ was given in 20%.\n\nThe SOFs spatially integrating the higher frequency signals (2 cpd) (F3) passed information through the windows which size was about 0.25 of the face (Figure 3A,C). As a result, the test images were formed, shown in Figure 3B,D. In this case the performance was again improved. The observers determined the gender in 94.3%.\n\nA further reducing of the SOFs‘ size while increasing the carrier frequency led only to deterioration of the performance (Figure 4, Figure 5, Figure 6).\n\nAll obtained results are summarized in the Table 1.\n\nIntegration of the information which was extracted by three SOFs from the low-frequency half of the spectrum (F1+F2+F3) (Figure 7A) did not improve the performance comparing with using only F3 (92.1% versus 94.3% respectively).\n\nBut this operation (F1+F2+F3) makes it to identify the person if the initial image is a familiar face25 (Figure 7B).\n\nA – the unfamiliar face from our experiment, B – the familiar face.\n\n\nDiscussion\n\nThe issue of ‘face perceiving’ can be divided conditionally into two parts: the allocation of the useful information (the reduction of redundancy) and the building a sizer of the selected information (the recognition). Our research concerns first part of this issue.\n\nAmong all the known algorithms of finding the ‘regions of interest’ only the small part could be viewed as neural26–32. These algorithms can be divided into the modular and the net. In case of the first ones the weight is given, in case of the second ones it is formed during the training of the net. The approach used by us in our work is based on the modular architecture of the earlier levels of processing, finishing with the automatic allocation of the useful information from an incoming image.\n\nIn our research the first order filters formed six copies of an incoming image with different definition, and the second order filters were used as windows, which were at the maximum level of difference from surroundings by contrast, orientation or spatial-frequency.\n\nThe received results show that the identification of a face is more effective on the carrier frequency 2 cpd. This conforms with the other authors’ data that showed that the identification of faces is faster and more precise if the frequencies of the middle range are used16,17,33–37. So what is new in our information?\n\nWe’ve shown that not the whole face is informative, but only its regions with spatial heterogeneity. Meaning, in task of detection of a human face and the definition of the gender information of a whole face is significantly redundant. It does not contradict with the data that processing of a human face is holistic38,39. It’s just that the integrated information concerning its most informative areas could be enough for the holistic description of it.\n\nIf 100% would be the sum of all second-order filters activated by our images we can presume that at frequency of 2pcd the volume of selected information would be 1%. Reminder, this amount of information is enough to determine gender confidently.\n\nTo identify a familiar face, determination of regions of interest on one of the frequencies is not enough. The summation of the low-frequency half of spectrum is necessary at least (Figure 8). High-frequency information is not crucial for the gender determination and identification, but useful in perceiving details and delicate differentiation.\n\nAt the left – Einstein, at the right – princess Diana.\n\nThus been said, we chose areas of image that differ most from the surroundings in contrast, orientation and spacial frequency to be the most informative. The second order filters that we used form maps of convexity for every carrier frequency. As a result we have the “embedded maps”. At the lowest of the used frequencies one of the filters selects face as a whole. The following maps select areas of a face that are smaller and smaller. If the object approaches or retreats, so that its size changes in a certain range, the embedded maps stay the same. The difference would be only if the object approaches the same regions of interest would be allocate by second-order filters, which are tuned at lower carrier frequency, and if it retreats – at the higher one.\n\nThe smaller the window, which transmits the information, the higher its definition is. As a result, the same portions of information are transmitted through every window. If size of an object or it’s turns is changing, general capacity and nature of information transmitted by second-order filters stays the same.\n\n\nConclusions\n\nNote that the information allocated with the given algorithm is useful for perceiving faces, the following hypothetical model of second-order filters image formating can be proposed. The face describing is simultaneous in a number of definition levels. At the relatively low level a face is described as a whole. With the higher definition transmits information concerning large objects of a face. Every higher level describes even smaller details. Wherein the given information allocates and transmits with parallel frequency channels. As a result, a hierarchical description of a face formed parallel, according to automatic algorithm. Wherein, the system of decision making can not use all available information. Elaborations will be made until specific visual task will be resolved.\n\nThe obtained results allow to assume that second-order filters are suitable candidates to the role of mechanism of convexity map formating, and information they allocate can be used to form a face image.\n\n\nData availability\n\nF1000Research: Dataset 1. Frequencies of different types of the responses, 10.5256/f1000research.5975.d4149940", "appendix": "Author contributions\n\n\n\nBabenko V.V. is the author of idea and method. He analyzed the obtained results and wrote the manuscript. 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Data Source" }	[ { "id": "7766", "date": "06 Mar 2015", "name": "Alexander Latanov", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript represents very interesting study on second-order visual filters that determine perception of face feature. These filters are associated with second-order neuronal populations that combine the outputs of striate neurons coding the primary visual features. The authors assume that second-order filters represent the mechanism of salience map formation. This map constitutes the basis for the recognition of human faces.I recommend this article for indexation.However the manuscript needs to be slightly corrected before indexation.What is the difference between abbreviation ‘cpi’ and ‘cpd’? Sometimes author use the term ‘spacial frequency’ and sometimes the term ‘spatial frequency’. It seems that the next two sentences represent the result of splitting of whole sentence: ‘Wherein, the system of decision making can not use all available information. Elaborations will be made until specific visual task will be resolved’.", "responses": [] }, { "id": "7885", "date": "09 Apr 2015", "name": "Talis Bachmann", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper includes several interesting ideas (e.g., how different response categories by subjects allow to know what is salient in the test image, using spatial heterogeneity areas as the basic image processing strategy). However, in its present form it could not be accepted by a standard peer review based scientific journal. Among several problems the following stand out.The language is poor; the manuscript should be thoroughly edited by a native speaker. Authors should carefully and convincingly persuade a reader why their study is valid for real face perception. As it stands now, they artificially create an image of some object (e.g., face) with spurious new visual characteristics, then they use these images for a perception task by human subjects and then they generalize for normal visual perception. It seems they do not study real perception of untransformed objects belonging to certain category, but study perception of specific artificially created stimuli. In the Introduction (e.g., first two paragraphs) needed specific references are absent.There are many minor points to be corrected (e.g., kd pro cd, Procedure too vaguely described, etc.)", "responses": [] } ]	1	https://f1000research.com/articles/4-10
https://f1000research.com/articles/4-7/v1	09 Jan 15	{ "type": "Study Protocol", "title": "Association between obesity and depression in patients with diabetes mellitus type 2; a study protocol", "authors": [ "Eduardo De la Cruz-Cano", "Carlos Alfonso Tovilla-Zarate", "Emilio Reyes-Ramos", "Thelma Beatriz Gonzalez-Castro", "Isela Juarez-Castro", "Maria Lilia López-Narváez", "Ana Fresan", "Eduardo De la Cruz-Cano", "Emilio Reyes-Ramos", "Thelma Beatriz Gonzalez-Castro", "Isela Juarez-Castro", "Maria Lilia López-Narváez", "Ana Fresan" ], "abstract": "Background: Diabetes mellitus and depression are highly prevalent conditions throughout the world and have significant impact on health outcomes. It has been estimated that diabetes mellitus type 2 affects about 246 million people in the world; nevertheless, incidence varies among countries. There is evidence that depression is associated with a poor metabolic control in patients with type 2 diabetes mellitus that present other health problems (such as hypertension and obesity). The aim of this study protocol is to determine if obesity increases the risk for depression in patient with diabetes type 2.Methods: The analysis will be reported following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA).The studies suitable for inclusion will be assessed by the Newcastle-Ottawa Scale (NOS) to determine their methodological quality. To identify the studies of interest, we will search on PubMed and EBSCO databases. We will use the following keyword combinations: \"Diabetes Mellitus type 2 AND obesity AND depression\", \"depression AND Diabetes Mellitus type 2\", \"Diabetes Mellitus type 2 AND body mass index cross sectional study\", \"depression AND obesity cross-sectional study\". Causes for exclusion will be publications that studied patients diagnosed with diabetes mellitus type 1; articles that focused on the treatment and complications of diabetes mellitus type 2; publications that have studied other clinical or psychiatric conditions (for instance, seizure disorder or history of schizophrenia, bipolar disorder, psychotic symptoms or dementia).Conclusion: The results of this study will form the basis for a better understanding of the association between obesity and depression in patients with diabetes mellitus type 2, and will allow development of prediction tools and better interventions. It is evident that several modifiable and non-modifiable risk factors play an important role in the pathogenesis of diabetes among population. Currently, evidence for the deleterious effects of diabetes mellitus type 2 are based on cross-sectional or other observational designs. Therefore, this study will have important implications for future research and public health guidance.", "keywords": [ "Diabetes Mellitus type 2", "obesity", "body mass index", "depression" ], "content": "Background\n\nDiabetes and depression are highly prevalent conditions throughout the world and have significant impact on health outcomes. Diabetes mellitus is a chronic-degenerative disease, characterized by high levels of blood glucose1–3. It has been estimated that diabetes mellitus type 2 affects about 246 million people in the world4; nevertheless, incidence varies among countries5,6. The International Diabetes Federation has anticipated an increase of 366 million people by 2030, giving a total of 552 million people with diabetes type 2 in the world6,7.\n\nThe diabetes type 2 is a complex disease, where hereditary and metabolic factors interfere8,9. Literature suggests there is a correlation between type 2 diabetes and mood alterations such as depression and neuropsychiatric disorders; for instance, major depressive disorder10,11, schizophrenia12,13, mild cognitive impairment14,15 and suicidal behavior16. It also has been observed that depression could cause an increase in all-cause mortality risk (approximately 70%)17; it is also the most common mental disorder and generates a great impact on people and society in terms of suffering, disability and economic costs, a phenomenon that seems to occur in many parts of the world; in this context, it has been reported that depression affects 350 million people worldwide18; for example, a research by Talbot et al. suggests that depression is not only a direct consequence of diabetes; depression may also be a risk factor for the onset of diabetes type 219. Patients with diabetes mellitus type 2, often present a careless attitude towards their disease, resulting in metabolic decompensation, with high and low glycemic levels which could trigger mood alterations20,21. Diabetes mellitus 2 is also associated with a higher risk of comorbid depression compared with the general population22. It has been suggested that diabetes type 2 could be conditioned by depression, anxiety or anguish23–25; nevertheless, the reason for this association is not clear26,27. The neurobiological mechanisms that could explain the association between depression and diabetes mellitus type 228 could include 1) the alterations involved in the metabolism of biogenic amines (serotonin and norepinephrine), from the adrenal-pituitary-hypothalamus axis (by increasing cortisol)28–30; 2) trophic agents such as Brain Derived Neurotrophic Factor (BDNF) through Glycogen Synthase Kinase-3 (GSK-3)31,32. The GSK-3 is a serine/threonine protein kinase that mediates the addition of phosphate molecules into serine and threonine amino acid residues. It consists of two isoforms, α and β33,34. It is possible that an over activation of GSK-3 play an important role in the pathogenesis of the development of schizophrenia and mood disorders such as bipolar disorder and major depression in patients with diabetes mellitus type 235,36. Furthermore, it has been suggested that the presence of metabolic alterations in patients with diabetes type 2 such as obesity, could increase the severity of depression37–40. The distinct mechanisms that link obesity to insulin resistance and diabetes mellitus type 2 are related to an increased production of adipokines and more adipose tissue as a result41,42; these molecules are involved in many clinical manifestations of diabetes mellitus type 2 and they are also associated with arterial hypertension and cardiovascular disease43. First, the adipose tissue of the obese patient becomes resistant to the action of insulin due to the effect of some of these adipokines; for instance, the tumoral necrosis factor alpha (TNF-α) and interleukine-6 (IL-6)44. Secondly, this resistance occurs in other tissues; therefore, insulin and glucose levels increase. This increase, along with high adipokines levels (that occur in diabetes mellitus type 2), lead to different adverse events, such as endothelial dysfunction45, increase in oxidative stress46, impairments in lipoprotein metabolism and increase in blood pressure47. For a review see Antuna puente et al.48. For example, a research by Svenningsson et al. suggests an association between depression and obesity in patients with diabetes mellitus type 2 in both genders; this study reported that at least one in five men and one in three women showed depression in diabetic type 2 patients with obesity49. Recently, a report showed that there is a positive association between having a high body mass index and the risk to develop diabetes mellitus type 250. In general, literature shows evidence that depression is associated with metabolic disorders in patients with type 2 diabetes mellitus27.\n\nIn this work we will focus on searching a correlation between obesity and depression in patient with diabetes type 2. The aim of this study is to determine if obesity in patients with diabetes type 2 increases the risk of depression.\n\nPICOT QUESTION: Does obesity increase the risk of depression in Diabetes Mellitus type 2 patients?\n\n\nMethods/Design\n\nThe study protocol will be conducted and reported in compliance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines51. In accordance with the guidelines, our study protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO)52, on 08 October, 2014 (registration number CRD42014014034).\n\n\nLiterature search strategy\n\nThe selection of publications and the reporting of results for the study protocol will be conducted in accordance with the PRISMA guidelines51. We will search on PubMed and EBSCO databases. We will further scan reference lists in relevant reviews and publications retrieved for the purpose of our study protocol. There will be no initial limit on the date of publication. We will use the following keyword combinations: \"Diabetes Mellitus type 2 AND obesity AND depression\", \"depression AND Diabetes Mellitus type 2\", \"Diabetes Mellitus type 2 AND body mass index AND cross sectional study\", \"depression AND obesity AND cross-sectional study\". The bibliography of the articles chosen will also be examined in order to find more articles that might not be on the electronic databases. We will only include case-control, cross-sectional and cohort studies. The planned procedure is illustrated in Figure 1.\n\n\nEligibility criteria\n\nTitles and abstracts will be screened for eligibility according to the following inclusion and exclusion criteria.\n\nFor the purpose of this study protocol will be included publications in English language that examined the relation of body mass index (BMI > 30 kg/m2) and severity of depression in patients with diabetes type 2.\n\nCauses for exclusion will be: publications that studied patients diagnosed with diabetes mellitus type 1; articles that focused on treatment and/or complications of diabetes mellitus type 2; publications or clinical trial that have focused on treatment of metabolic and psychiatric disease (for instance, mood stabilizers, neuroleptic, antidepressant, benzodiazepines, seizure disorder, history of schizophrenia, bipolar disorder, psychotic symptoms and dementia).\n\n\nType of studies\n\nThis study protocol will include case-control, cross-sectional and existing cohort studies up to date.\n\n\nType of participants\n\nThe participants will be adults (aged 18 years and over), diagnosed with diabetes mellitus type 2. For the purpose of this review, only overweight and obese type 2 diabetes mellitus patients with symptoms of depression will be included.\n\n\nScreening\n\nFirst, Two independent reviewers will read the titles of all the citations retrieved from the electronic database searches and removed all citations that are clearly not related to our study. Next, the abstract will be assessed to determine if the study satisfies the inclusion criteria. If from the abstract it is unclear whether the selection criteria are met or not, the full article will be scanned. Any discrepancy for inclusion will be discussed with a third or fourth author. Once the appropriate articles have been chosen for further analysis, two or three authors (independently) will be involved in the assessment of each article and data extraction. Further studies may be excluded as a result of not being relevant for our study. Further studies may be included through searching the reference lists in publications selected for the review. All the studies included will be read in detail and the relevant information extracted. The degree of agreement between the observers will be calculated by the Kappa coefficient; the studies that cause disagreement will be reviewed again, then the observers will decide the inclusion/exclusion together. The studies selected will be evaluated for quality and incorporation of gender perspective. Studies deemed for inclusion will be scored for methodological quality using the Newcastle-Ottawa Assessment Scale (NOS)53. Results will be analyzed using a narrative synthesis. To give more support to our analysis, we will consider the GRADES scale procedures (http://www.gradeworkinggroup.org).\n\n\nAnalysis of results\n\nA descriptive synthesis of important characteristics will be undertaken independently, including, author, year study, sample characteristics, type of study design, length of follow-up (for cohort studies), exposure variable characteristics, dependent variable characteristics, method used to ascertain diabetes status and body mass index; assessment of depression, relative risk or equivalent associated with diabetes mellitus type 2 and obesity. A quantitative synthesis of effects will not be attempted because of substantial methodological heterogeneity among studies.\n\nWhenever possible, adjusted relative risk (RR) or equivalent and associated 95% CI will be extracted directly from studies. For studies that present RR by subgroups (for example, relative risk associated with Body Mass Index, kg/m2 ≥30) the data for each subgroup will be additionally extracted. Authors will be contacted via email for any missing relevant information. Also, data will be analyzed descriptively. The systematic review will be presented in tables comparing quality measurements and the data previously mentioned.\n\n\nDiscussion\n\nThe aim of this study protocol is to verify if there is a direct relation between depression and obesity in patients diagnosed with Diabetes Mellitus type 2, with the aim of improving the treatment of these patients, through an updated and quantitative estimate of the risk of depression associated with obesity. This study protocol will include a wide number of study designs; therefore, a subgroup analysis will be performed, to understand the relation between depression and obesity in patients with type 2 diabetes according to type of study. Furthermore, literature suggests that age is associated with depression, as well as other emotional alterations54,55; therefore, age could also influence patients with obesity to develop depression. Nevertheless; up to today, there are no-systematic reviews that search for this association. It is important to know if there is a connection between a high body mass index (BMI) and emotional alterations of diabetes mellitus type 2 patients56,57. Finally, depression and obesity appear to be linked with poorer behavioral management of diabetes and glycemic control; therefore, the need for comprehensive interventions worldwide that target depression in conjunction with the type 2 diabetes mellitus management. The findings from this study protocol will be widely disseminated through discussions with stake-holders, publication in a peer-reviewed journal and a conference presentation. This study protocol on diabetes and depression will bring to light knowledge gaps in the area and will offer directions for future researches.\n\n\nAbbreviations\n\nBDNF: Brain Derived Neurotrophic Factor; BMI: Body Mass Index; CRH: Corticotropin-Releasing Hormone; GSK-3: Glycogen Synthase Kinase-3; IL-6: Interleukine-6; NOS: Newcastle-Ottawa Scale; PRISMA: preferred reporting items for systematic reviews and meta-analyses; PROSPERO: Prospective Register of Systematic Reviews; RR: risk ratio; TNF-α: Tumoral Necrosis Factor alpha; T2DM: Type 2 Diabetes Mellitus.", "appendix": "Author contributions\n\n\n\nEC was responsible for the formulation of the research question, design of protocol, drafting the manuscript and was also responsible for the manuscript submission and responding to the reviewer comments. EC, CT, ER, and TBG - assisted with the formulation of the research question, design of protocol and commented on the manuscript drafts. MLL and AF commented on the manuscript drafts. IJ assisted on the formulation of the research question, design of protocol and commented on the manuscript drafts. All authors read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study protocol presents independent research. The views expressed in this publication are those of the author(s); therefore this study has not received funding.\n\n\nReferences\n\nAlharbi KK, Khan IA, Munshi A, et al.: Association of the genetic variants of insulin receptor substrate 1 (IRS-1) with type 2 diabetes mellitus in a Saudi population. Endocrine. 2014; 47(2): 472–477. PubMed Abstract \| Publisher Full Text\n\nGanasegeran K, Renganathan P, Manaf RA, et al.: Factors associated with anxiety and depression among type 2 diabetes outpatients in Malaysia: a descriptive cross-sectional single-centre study. BMJ Open. 2014; 4(4): e004794. 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PubMed Abstract \| Publisher Full Text\n\nSacco WP, Wells KJ, Vaughan CA, et al.: Depression in adults with type 2 diabetes: the role of adherence, body mass index, and self-efficacy. Health Psychol. 2005; 24(6): 630–4. PubMed Abstract \| Publisher Full Text\n\nLabad J, Price JF, Strachan MW, et al.: Symptoms of depression but not anxiety are associated with central obesity and cardiovascular disease in people with type 2 diabetes: the Edinburgh Type 2 Diabetes Study. Diabetologia. 2010; 53(3): 467–471. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJulia C, Czernichow S, Charnaux N, et al.: Relationships between adipokines, biomarkers of endothelial function and inflammation and risk of type 2 diabetes. Diabetes Res Clin Pract. 2014; 105(2): 231–238. PubMed Abstract \| Publisher Full Text\n\nde Luis DA, Aller R, Izaola O, et al.: Role of insulin resistance and adipocytokines on serum alanine aminotransferase in obese patients with type 2 diabetes mellitus. Eur Rev Med Pharmacol Sci. 2013; 17(15): 2059–2064. PubMed Abstract\n\nZiegler D: Type 2 diabetes as an inflammatory cardiovascular disorder. Curr Mol Med. 2005; 5(3): 309–322. PubMed Abstract \| Publisher Full Text\n\nPereira FO, Frode TS, Medeiros YS: Evaluation of tumour necrosis factor alpha, interleukin-2 soluble receptor, nitric oxide metabolites, and lipids as inflammatory markers in type 2 diabetes mellitus. Mediators Inflamm. 2006; 2006(1): 39062. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBachmayer C, Kemmer A, Ehrmann N, et al.: Adipokines and endothelial dysfunction in obesity WHO°III. Microvasc Res. 2013; 89: 129–133. PubMed Abstract \| Publisher Full Text\n\nCrujeiras AB, Díaz-Lagares A, Carreira MC, et al.: Oxidative stress associated to dysfunctional adipose tissue: a potential link between obesity, type 2 diabetes mellitus and breast cancer. Free Radical Res. 2013; 47(4): 243–256. PubMed Abstract \| Publisher Full Text\n\nAdler AI, Stratton IM, Neil HA, et al.: Association of systolic blood pressure with macrovascular and microvascular complications of type 2 diabetes (UKPDS 36): prospective observational study. BMJ (Clinical research ed.). 2000; 321(7258): 412–419. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAntuna-Puente B, Feve B, Fellahi S, et al.: Adipokines: the missing link between insulin resistance and obesity. Diabetes Metab. 2008; 34(1): 2–11. PubMed Abstract \| Publisher Full Text\n\nSvenningsson I, Björkelund C, Marklund B, et al.: Anxiety and depression in obese and normal-weight individuals with diabetes type 2: a gender perspective. Scand J Caring Sci. 2012; 26(2): 349–354. PubMed Abstract \| Publisher Full Text\n\nGanz ML, Wintfeld N, Li Q, et al.: The association of body mass index with the risk of type 2 diabetes: a case–control study nested in an electronic health records system in the United States. Diabetol Metab Syndr. 2014; 6(1): 50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoher D, Liberati A, Tetzlaff J, et al.: Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. PLoS Med. 2009; 6(7): e1000097. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBooth A, Clarke M, Dooley G, et al.: PROSPERO at one year: an evaluation of its utility. Syst Rev. 2013; 2(1): 4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStang A: Critical evaluation of the Newcastle-Ottawa scale for the assessment of the quality of nonrandomized studies in meta-analyses. Eur J Epidemiol. 2010; 25(9): 603–605. PubMed Abstract \| Publisher Full Text\n\nKaton WJ, Simon G, Russo J, et al.: Quality of depression care in a population-based sample of patients with diabetes and major depression. Med Care. 2004; 42(12): 1222–1229. PubMed Abstract\n\nKendrick T, Dowrick C, McBride A, et al.: Management of depression in UK general practice in relation to scores on depression severity questionnaires: analysis of medical record data. BMJ (Clinical research ed.). 2009; 338: b750. PubMed Abstract \| Publisher Full Text\n\nGudelj-Radi J, Davidović D, Avramović D, et al.: [Factors mediating the depression in the adult obese outpatients]. Srp Arh Celok Lek. 2007; 135(1–2): 61–66. PubMed Abstract \| Publisher Full Text\n\nSacco WP, Wells KJ, Friedman A, et al.: Adherence, body mass index, and depression in adults with type 2 diabetes: the mediational role of diabetes symptoms and self-efficacy. Health Psychol. 2007; 26(6): 693–700. PubMed Abstract \| Publisher Full Text" }	[ { "id": "7266", "date": "16 Jan 2015", "name": "Hans-Peter Volz", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the study plan of De la Cruz-Cano and co-workers the parameters for a structured literature research about the relationship between diabetes type II, obesity and diabetes are reported. The rational for this planned investigation is stated very clearly, the methods proposed seem adequate, the purpose of the planned investigation is highly welcomed.Therefore the study protocol should be indexedin order to stimulate the discussion about this very important topic and the planned literature review.", "responses": [] }, { "id": "7781", "date": "13 Mar 2015", "name": "Janusz Rybakowski", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe aim of this paper is to make a systematic review and meta-analysis of the association between obesity and depression in patients with diabetes mellitus type 2. The results of the study may allow development of prediction tools and better interventions in such condition. The rationale for the study is provided and the methods proposed seem correct.My only reservation is that, in psychiatric patients and in general population, obesity has been found to be mostly associated with a specific kind of depression, i.e. depression with atypical features or atypical depression ( Levitan et al, 2012, Glaus et al, 2013, Chou and Yu, 2013). Therefore, I would suggest that the authors may take this kind of depression into account in their analysis.", "responses": [] } ]	1	https://f1000research.com/articles/4-7
https://f1000research.com/articles/3-291/v1	28 Nov 14	{ "type": "Opinion Article", "title": "Shaping the Future of Research: a perspective from junior scientists", "authors": [ "Gary S. McDowell", "Kearney T. W. Gunsalus", "Drew C. MacKellar", "Sarah A. Mazzilli", "Vaibhav P. Pai", "Patricia R. Goodwin", "Erica M. Walsh", "Avi Robinson-Mosher", "Thomas A. Bowman", "James Kraemer", "Marcella L. Erb", "Eldi Schoenfeld", "Leila Shokri", "Jonathan D. Jackson", "Ayesha Islam", "Matthew D. Mattozzi", "Kristin A. Krukenberg", "Jessica K. Polka", "Gary S. McDowell", "Kearney T. W. Gunsalus", "Drew C. MacKellar", "Sarah A. Mazzilli", "Vaibhav P. Pai", "Patricia R. Goodwin", "Erica M. Walsh", "Avi Robinson-Mosher", "Thomas A. Bowman", "James Kraemer", "Marcella L. Erb", "Eldi Schoenfeld", "Leila Shokri", "Jonathan D. Jackson", "Ayesha Islam", "Matthew D. Mattozzi" ], "abstract": "The landscape of scientific research and funding is in flux and affected by tight budgets, evolving models of both publishing and evaluation, and questions about training and workforce stability. As future leaders, junior scientists are uniquely poised to shape the culture and practice of science in response to these challenges. A group of postdocs in the Boston area who are invested in improving the scientific endeavor, planned a symposium held on October 2nd and 3rd, 2014, as a way to join the discussion about the future of US biomedical research. Here we present a report of the proceedings of participant-driven workshops and the organizers’ synthesis of the outcomes.", "keywords": [ "biomedical research", "funding", "training", "publishing" ], "content": "Executive summary\n\nThe Future of Research Symposium, held in Boston in October 2014, was born out of a desire on the part of junior scientists to influence discussions about the future of biomedical research in the United States. Current trainees in academic research represent a talented pool of people contributing to scientific progress. This pool, however, is far larger than the current system is able to support in the long term. As structural forces governing the funding and administration of science push many graduate students and postdocs out of research, the public funds supporting their training are poorly repaid.\n\nWe view the current policy makers’ reluctance to invest in science as a short-sighted and potentially catastrophic mistake. Furthermore, the current distribution of funding priorities and the way that funding agencies, universities, and journals reward scientists leads directly to inefficiencies within the conduct of research. While scientists continue to advocate for increased funding, they must also create a scientific enterprise that is sustainable with the current resources. A sustainable long-term investment in science, and an appreciation of the young people who carry it out, are essential to the long-term economic and social interests of the US. Specifically, the hyper-competition that we have all experienced, which stunts scientific curiosity and productivity, breeds fabrication and carelessness in the publication of data, and leads to a waste of valuable resources and intellectual capital, must be alleviated. In all of our discussions, we have kept two goals in mind: maximize the potential for wide-ranging and fundamental scientific discovery; and minimize the loss of talented young researchers who can contribute greatly to science.\n\nIn addition to voicing our concerns, we junior scientists recognize that we need to become more aware of the issues facing the research enterprise, comprised of academia, industry, publishing, and government. To accomplish this, the initial sessions of the symposium consisted of a series of talks and panel discussions from leaders who have been outspoken about the challenges that science faces. These were followed by workshops designed to elicit the opinions and ideas of participants, largely postdocs and graduate students, on problems and solutions surrounding training, the structure of the research workforce, funding, and incentives and rewards in science. We present the outcomes of those discussions in this report, which represents a united voice of young biomedical scientists, conveying our concerns about the sustainability of the research enterprise and our hopes for change.\n\nFrom the many ideas presented in the workshops and continued discussions among the organizers, we have distilled the following three principles to guide future activities towards scientific reform:\n\nWe recommend increased connectivity among junior scientists and other stakeholders to promote discussions on reforming the structure of the scientific enterprise.\n\nWe advocate for increased transparency. This includes the number and career outcomes of trainees, as well as the expectations of the balance between employment and training in individual postdoctoral appointments.\n\nWe call for an increased investment in junior scientists, with increased numbers of grants that provide financial independence from Principal Investigator (PI) research grants, and increased accountability for the quality of training as a requirement of funding approval.\n\nJunior scientists must take a larger role in engaging with these issues. As the engine of academic research, junior scientists must be given a voice fitting their role as major stakeholders in the scientific enterprise. Equally, junior scientists must be educated about their role so that they have the context necessary to make a well-informed contribution and to effectively advocate for their interests. By bringing our concerns into the conversation that guides policy, the dialogue will be enriched with diversity and fresh perspectives. We encourage our peers to continue this conversation, engage their colleagues, and to get involved in shaping the Future of Research.\n\n\nGenesis of the Future of Research Symposium\n\n““The government should provide a reasonable number of undergraduate scholarships and graduate fellowships in order to develop scientific talent in American youth. The plan should be designed to attract into science only that proportion of the youthful talent appropriate to the needs of science in relation to the other needs of the nation’s high priority”. And I think that is one of the places where we have in biomedical science gone astray”.\n\nShirley Tilghman, quoting Vannevar Bush, at a meeting of the President’s Council of Advisors on Science and Technology (PCAST), September 19 2014, (“PCAST Meeting 2014”, 2014).\n\nA large portion of the nation’s science and engineering research is carried out by graduate students and postdocs. Because of this, the current culture of training places a heavy emphasis on research and publications, at the expense of “soft skill acquisition” or career development.\n\nIn the US, pre-doctoral training in the biomedical sciences takes 6.5 years on average (Figure 3 of (Biomedical Research Workforce Working Group, 2012)), and includes research experience culminating in a PhD dissertation. This process is overseen by a committee of 3–5 faculty members and requires the development of some core skills.\n\nIn contrast, it is notoriously difficult to determine how many postdoctoral scholars there are, let alone what kind of training they are or should be receiving. The National Institutes of Health (NIH) and the National Science Foundation (NSF) define a postdoctoral scholar as “an individual who has received a doctoral degree (or equivalent) and is engaged in a temporary and defined period of mentored advanced training to enhance the professional skills and research independence needed to pursue his or her chosen career path” (Bravo & Olsen, 2007). Most postdoctoral “trainees” conduct research under the supervision of a single Principal Investigator (PI), and there are no explicit guidelines to determine what training a postdoc should receive or when this training is complete. In reality, postdoctoral research is often not a training period at all, but a time when experienced junior researchers contribute significantly to the goals of a PI’s grant. There is no expectation of specific training, and no defined period in which the training takes place: “training” ends only when the postdoc takes another job.\n\nIn spite of the number of years spent in pre- and postdoctoral training, only a handful of scientists feel that they are adequately prepared for any job other than conducting research. Many feel they are unaware of what jobs they should be training for, let alone what skills those jobs require. One common complaint is that scientists are not being prepared for non-faculty positions, yet the number of new faculty who are unprepared for their non-research responsibilities (such as managing employees and budgets or teaching) suggests that graduate students and postdocs are not even being properly trained to become future faculty.\n\nWhile the number of US graduate students in biomedical science have increased from about 46,500 in 1993 (Table B-18 in (National Science Foundation, 1994)) to almost 71,000 in 2012 (Table 16 in (National Science Foundation, 2014)), the fraction of PhDs in life sciences in a tenure-track position 5 years post-PhD decreased from 17.3% (1993) to 10.6% (2010) (Table 3–18 in (National Science Board, 2014). There has also been a tremendous shift in the job market outside of academia over the past decades, with a general slowdown and even contractions in government and industry. This situation has long been deemed unsustainable by many senior academics (Bourne, 2013a; Stephan, 2012a; Stephan, 2012b; Teitelbaum, 2008).\n\nWith the number of graduate students increasing faster than the number of faculty positions (Figure 1 in (Schillebeeckx et al., 2013)), it is unsurprising that the NIH estimates that the number of postdoctoral researchers also doubled during that time. However, estimates of the number of postdocs vary drastically. The National Research Council puts the number of postdocs at just over 50,000 (National Research Council (US) Committee to Study the National Needs for Biomedical, Behavioral, and Clinical Research Personnel, 2011), but the NIH states that this could be under-estimated by as much as a factor of two (Biomedical Research Workforce Working Group, 2012). According to a recent report by the National Postdoctoral Association (NPA), the NPA’s 167 member institutions alone estimate that their postdoc offices serve about 79,000 postdocs (Ferguson et al., 2014).\n\nData from the NSF Survey of Doctorate Recipients suggests that the US-trained biomedical PhDs “who do the longest postdocs are the ones who go on to tenure-track academic research careers” (Rockey, 2012). However, in spite of the number of scientists remaining in long postdocs in the hopes of landing a tenure-track faculty position, the data show clearly that academia is an “alternative” career, not the default. In 2010, less than 15% of US-trained science, engineering and health sciences postdocs had obtained a tenure-track faculty position within 5–7 years of completing their PhD (Sauermann & Roach, 2012). The rest of the job market encompasses many fields that are expanding and can benefit from the trained minds of PhDs and postdocs. These include (but are not limited to): consulting for life sciences, biotech and biopharmaceutical industries, sales and marketing of technologically advanced products, regulatory affairs, science policy, science communications, and intellectual property.\n\nEven though the majority of postdocs will do something other than become tenure-track faculty members, the default assumption of many PIs (and their mentees) remains that graduate students and postdocs will follow their mentors’ career trajectory and acquire an academic faculty position at a research-intensive institution. The data show that by the end of their PhD training, only 50% of graduate students want to become academics, and that expectations change over time: a faculty position becomes less attractive over the course of a PhD, in spite of active encouragement by advisors (Sauermann & Roach, 2012).\n\nThus, many junior scientists want, and most will obtain, non-faculty jobs. However, few young scientists and their faculty mentors know what careers are actually available, let alone what skills those jobs require or how to obtain them. The mismatch between scientists’ career expectations and the realities of the job market has led to extended occupancy of postdoc positions and highly inflated expectations from academic employers for prior productivity.\n\nIn the US, the funding system has had a profound impact on the structure of universities and academic and applied research departments, and how the time of principal investigators and young scientists is spent.\n\nAs early as 2003, the rapid increase in funds over the previous decade was generating questions about where trainees would end up in the absence of a concomitant increase in academic positions (Russo, 2003). In response to these concerns, there have been calls for institutions to become more responsible for funding “hard-money” faculty positions, and to increase NIH incentives for doing so, rather than relying on external sources of funding for “soft-money” positions (Alberts, 2010). These problems were left unresolved, however, and now that there has been a contraction in funding they have become immediate. For institutions and individual researchers attempting to make long-term decisions, financial uncertainty makes planning very challenging. It is clear that simply putting more money into the system would provide only a temporary fix, not a long-term solution to the systemic problems with academic research (Alberts et al., 2014; Martinson, 2007).\n\nAn assumption of many industries is that increased competition between groups or individuals yields largely beneficial results. However, academic science in the US was essentially founded on Vannevar Bush’s principle of the “supreme importance of affording the prepared mind complete freedom for the exercise of initiative” (Bush, 1945). These two principles are incompatible.\n\nIndeed, we believe that the problems caused by the current unsustainable workforce are threatening the very foundations of scientific research. The high stakes and low expectations of success prevalent throughout biomedical research, from grant applications to hiring decisions, promote academic dishonesty (Lang, 2013). Also, success in grant applications and career progression relies heavily on publications (van Dijk et al., 2014). This can lead to hyper-competition for “high-impact” publications and in some recent cases, a lack of truth in publishing (Nosek et al., 2012; Sovacool, 2008). Competition also encourages scientists to present data in the most optimistic light, and to include only data that lead to a clean and understandable conclusion. As postdocs, we see and experience these pressures first-hand. The pressure to publish needs to be balanced with incentives for rigorous and honest scientific communication.\n\nHowever, dishonesty is not the only problem threatening the integrity of academic literature. Part of the scientific endeavor is to provide checks and balances, reproduce results, and highlight when reproducibility fails. However, it is difficult (and unrewarding) to publish the results of replicative experiments or negative data, and there is a worrying trend in the lack of reproducibility in some forms of analysis; this issue was recently highlighted with regard to the widely-used technique of fluorescence-activated cell sorting (Hines et al., 2014). Some journals have made a call specifically for papers reporting negative data, and there are indications that the NIH may be looking to drive more studies testing whether data can be reproduced (Collins & Tabak, 2014).\n\nHyper-competition can also discourage creative thinking and risk-taking, strong foundations of the scientific endeavor (Alberts et al., 2014). Rather than grant applications for innovative, breakthrough science, we have observed that hyper-competition results in “safe” applications, driving incremental, slow improvements on existing knowledge. It blunts the blade of science, preventing it from piercing through existing ideas and paradigms to expose new frontiers.\n\n\nJunior scientists must join the debate\n\nA range of problems with the biomedical research system in particular have been the subject of increasing alarm in the scientific community (Alberts et al., 2014; Bourne, 2013a; Bourne, 2013b; Bourne, 2013c). While the focus has mostly been on US academic science, many of the problems are universal. These issues are not just relevant to those inside academia: due to their importance to national competitiveness, they are increasingly featured in the popular media as well (Harris, 2014a; Harris 2014b; Harris 2014c; Harris 2014d).\n\nThe public debate surrounding these issues has so far been led by senior members of academia (Alberts et al., 2014). One group that has yet to contribute significantly to the discussion is the largest group of researchers affected: graduate students and postdocs. Boston-area postdocs organized the Future of Research Symposium to raise awareness of the difficulties faced by young scientists and to provide a venue for further discussion and problem-solving during a set of interactive workshops.\n\nWe issued a call-to-arms to our peers to announce what we were doing, and to emphasize our view that young researchers should have a say in shaping the future direction of the research endeavor (McDowell et al., 2014a). To achieve our goal of giving a voice to the aspirations of young researchers, we synthesized the current issues that have been identified as obstructing the progress of scientific research into four focus areas: funding for biomedical research, training of the scientific workforce, the structure of the workforce, and incentives and rewards for scientists (McDowell et al., 2014c). Interactive problem-solving workshops honed in on each topic to explore the problems and propose solutions with the aim of formulating a response that we can provide to the larger scientific community. This document is the first to begin disseminating that response to foster and foment further discussion and action. Here we present the problems identified and tentative solutions suggested by participants in the workshops. We then discuss areas identified through ongoing discussions as requiring the most urgent action from young scientists to improve the Future of Research.\n\n“To be creative…emphasize new possibilities by disclosing those hidden episodes of the past when, even if in brief flashes, people showed their ability to resist, to join together, occasionally to win”.\n\nHoward Zinn (Zinn, 2014)\n\n\nSurvey of participants prior to the symposium\n\nIn order to focus the aims of the workshops, participants were invited to complete an anonymous survey of their ideas about how science should be conducted and supported, and the problems they identified with the current system. In all, 409 people responded to the survey, although not all offered a response to all questions (raw data are available in Appendix 1). Respondents were primarily postdocs and graduate students, but also included administrators, faculty, industry, research assistants and undergraduates (Figure 1). The survey included five short-answer questions; while these responses are not amenable to quantitative analysis, we have summarized them below.\n\nAnswers focused on several key points, listed here by the frequency with which they were mentioned, starting with the most commonly cited problems.\n\nIn the US, funding for basic science is inadequate to support long-term economic growth.\n\nThe quality of the scientific results being produced is compromised by the current structure of research funding and execution.\n\nThe research environment at present selects for proficiency at securing funding and publishing high-profile positive results, rather than rewarding scientific skepticism, curiosity, and balanced presentation of sometimes complex results.\n\nThe current funding system is unnecessarily bureaucratic and insufficiently transparent, reflecting temporary political whims, and the duration of NIH grants is too short to support the lengthy explorations necessary to accomplish truly novel, beneficial basic research.\n\nThe number of enthusiastic scientists competing for scarce funding encourages counter-productive levels of competition.\n\nExisting publication models exacerbate the problems arising from the inefficiencies of funding and the promotion of talent; journals disseminate research results in a periodic, page-limited manner that is outmoded in the internet era.\n\nThere were many concerns about mentorship, trainee freedom and related issues indicative of an imbalance between the supply of qualified scientists and the demand for sufficiently-funded basic research positions.\n\nThe most common responses to this question (most commonly mentioned first) included:\n\nCollaboration, meaning interdisciplinary teamwork between scientists in different institutions and fields, as well as across boundaries of status and seniority.\n\nOpenness in data, reagents, and evaluation of each other’s work.\n\nIntegrity and ethical research practices, innovation, and risk-taking.\n\nCritical thinking in the reporting and reproducibility of results.\n\nGreater outreach to the public to improve non-scientists’ awareness of the most crucial results in recent research.\n\nGreater efficiency in the research process, as well as entrepreneurship, academic-industry partnerships, and more effective measurement of training and outcomes in basic research.\n\nThe overall consensus from responses to this question focused on the importance of teaching scientists how to solve problems with scientific methods in an ethical fashion.\n\nTraining should be consistent across institutions, be multidisciplinary, and be independent of the race, sexual orientation, gender, gender identity or expression, national origin or cultural identification of its participants, to promote a community of diverse intellects.\n\nMentorship should involve close interactions between mentor and mentee and should include well-defined expectations for both parties.\n\nA common request was for job security amongst researchers. Suggestions for implementation included a restriction upon the total number of PhDs awarded, an expectation of retirement based upon the age of PIs, an increase in the number of staff scientist positions supported by federal research funds, and more rigorous evaluation of scientists across institutions, from the undergraduate to the principal investigator level.\n\nRespondents replied that government funding should balance an interest in both the long-term (basic) and short-term (applied) benefits of science.\n\nIndustrial/commercial entities should assume responsibility for the advances that are most directly commercializable, while federal funding should address projects that are more prospective.\n\nGovernment funding should support public health and environmental health research that is otherwise not addressed by the immediate, private concerns of individual donors.\n\nTo help support long-term research, some grants could be awarded to institutions, rather than individuals, to allow a community of researchers to decide among themselves which projects they find most meritorious.\n\nWithin basic research, funding outcomes should be independent of expectations of immediate profitability. Many crucial advances within science have been made based on open-ended inquiry, driven by the curiosity of the individual personalities involved, and these contributions have subsequently proven essential to technical innovation.\n\nRespondents also noted that excessive competition hinders collaboration and encourages non-productive duplication of experimental effort on select “hot topics”. The competition among qualified personnel for independent jobs is also highly inefficient in terms of wasted human capital.\n\nOnly 13% of graduate students, 16% of postdocs, and 18% of faculty respondents suggested that the workforce should not adapt to the existing funding trends. Of those opposed to adaptation, established researchers (faculty) considered it more important to ignore fluctuations in funding. Most respondents suggested adaptation, using varying strategies. Several faculty respondents focused their attention upon lobbying congress and turning to public outreach in order to convince our fellow citizens of the importance of funding basic biomedical research. Other suggestions included:\n\nReaching out to alternative funding sources, including state, local, and non-profit donors.\n\nInstituting longer timelines for approved grants.\n\nMore direct funding by universities for employee researchers, encouraging smaller labs with more direct PI-trainee oversight.\n\nGreater understanding that non-academic careers are actually the major outcome for PhD holders, and support and encouragement for students and trainees who enter such careers.\n\nMore transitional funds for entrepreneurial research and private-public partnerships.\n\nChanging the ratio of academic lab personnel between grad students, postdocs, technicians, and senior scientists.\n\nThe responses to this question were predominantly in favor of reducing the number of trainees per permanent position available in basic research, to steer funds towards more permanent positions, to seek alternatives to traditional funding sources (including private and nonprofit sectors), and to encourage greater regulation at the institutional and lab levels to address the efficiency of spending relative to the scientific research benefit produced.\n\nOverall, the respondents’ concerns and criticisms centered on a few key themes; however, there was disagreement regarding which issues are most important to the future of groundbreaking and sustainable science. We considered these suggestions indicative of a general dissatisfaction with the current research paradigm, but not necessarily prescriptive of specific and comprehensive solutions. The output of this survey is informative in gauging the general opinion of educated, disciplined, and curious people pursuing science in the US. Practical adjustments to academic science were discussed in the workshops, described in the following sections.\n\n\nParticipant-led Workshops at the Future of Research Symposium\n\nWorkshops were designed to allow participants to discuss issues identified as obstructing the progress of scientific research. Each workshop was overseen by three to four moderators from the organizing committee who provided some background on the current system and posed the specific objective for each session. The four objectives were to ask:\n\nHow can trainees be better prepared for careers in science in 2014?\n\nHow should the supply of postdocs and graduate students be matched to the demand for jobs in order to create a sustainable workforce?\n\nHow can the funding of academic research be structured to promote desired outcomes such as the discovery of basic knowledge, finding applications of knowledge for the betterment of society, and training the next generation of scientists?\n\nHow can the current system of incentives be fixed so that scientists and institutions are rewarded for the behaviors that are believed to support good science?\n\nWorkshops were broken down into two separate 90-minute sessions. The number of participants per topic per session was typically between 20 and 30. Individual participants were asked to write down the perceived problems with the current system on post-it notes and to post them on the wall. Working as a group, participants categorized these individual responses and identified major themes. Participants were then asked to individually write down possible solutions to the identified problems. This was once again done on post-it notes. Solutions were categorized according to the level of implementation, ranging from actions that can be accomplished by individual graduate students and postdocs to those requiring action from society as a whole. If time permitted, participants voted on solutions they found most compelling and discussed the pros and cons of these solutions further. Generally, there was not sufficient time to discuss any potential solutions in depth. We view these sessions primarily as a way to begin debate, not to end it.\n\nThe workshops identified a large number of problems and potential solutions, many of which were raised repeatedly, though the immediate topic of conversation varied. In the following sections, we present lists of proposed solutions, without necessarily endorsing each possible solution, together with a few common themes distilled from each workshop. The raw data for each workshop can be found in Appendices 2A–D.\n\n\nTraining for careers in science in 2014\n\nParticipants identified problems with the current training system in the following key areas (Appendix 2A):\n\nCulture of academia-focused training: The prevailing view of training focuses heavily on academia, where few scientists can obtain positions. This creates a sense of failure for those leaving academia.\n\n“[Young scientists have the] feeling there is no way to exit [academia] positively”.\n\nAbsence of awareness of non-academic job opportunities: Scientists have limited knowledge of careers outside of academia that require scientific training. They are not aware of the kinds of jobs they may be qualified for; the skills these different jobs may require; and how to successfully apply for these jobs.\n\n“[Scientists are] unaware that careers in science exist (outside of academia)”.\n\nPIs are not equipped to advance their mentees’ careers: PIs have little incentive to act as a mentor for a trainee’s career development, and limited training that would make them competent to do so.\n\n“For a lot of mentors, it’s not a priority to engage in your career path”.\n\nInformal training leads to inconsistent training: There is a lack of standardized training for any scientific career, be it academic or non-academic. PIs require multiple skills learned only from experience; current training was described as “spotty” and “overly specialized”. Training standards are highly variable between institutions and research groups.\n\n“Training is not formalized (expected to pick up stuff along the way)”.\n\nLack of professional skills training: Current training fails to teach skills that can be applied to both academic and non-academic careers, including people management, networking, writing, and presentation skills. Scientists learn to conduct research, but not to manage a research group.\n\n“Lack of “real world” professional skills”.\n\nLittle or no training on transitioning to industry: There is a dearth of training about how to transition from academia to industry. There are too few internship programs providing experience in industry.\n\n“You need to know someone in industry to get a job there”.\n\nIndividual graduate students and postdocs\n\nGraduate students and postdocs can identify the skills they need to develop (such as via the my Individual Development Plan (myIDP) tool (Fuhrmann et al., n.d.)), then collaborate with each other and with graduate programs and postdoctoral offices to acquire training.\n\nPostdocs should advocate for themselves, network with each other, and provide mentorship to each other.\n\nPIs and research groups\n\nWe must correct the misconception that all scientists will pursue an academic career.\n\nPIs should allow time for career development; recent data suggests this will not detract from research productivity (Rybarczyk et al., 2011; Strategic Evaluations, Inc., 2014).\n\nInstitutions\n\nInstitutions should make adequate, appropriate training available and insist that PIs allow attendance. “Adequate, appropriate training” should enhance the professional skills that graduate students and postdocs have identified as important for their chosen careers.\n\nInstitutions should develop teaching and industry opportunities.\n\nInstitutions could create networks that allow for past, current and future trainees to communicate about careers.\n\nFunding agencies and the scientific community\n\nAvailability of adequate, appropriate training should be mandated across all institutions.\n\nGrant incentives should be used to encourage PIs to facilitate adequate training.\n\nThe current culture of training places heavy emphasis on research and publications, leaving little time for “soft skill” or career development. Postdoctoral “training” is a misnomer: as one participant put it, “If you’re going to call me a trainee, then train me”.\n\nRather than force everyone to be trained for the same (academic) career path, institutions should provide opportunities for trainees to acquire skills that are useful in multiple career paths, and PIs should be required to allow trainees access to these training opportunities.\n\nPostdocs were consistently called “the lost people” and “the invisible people”. Postdocs do not yet have a coherent voice, and we must change this. Postdoctoral associations should be advocating for access to training, both in provision and time allowance, in their institutions. The National Postdoctoral Association should have a stronger voice in advocating for postdoctoral training at a national level. Trainees should involve themselves with their learned societies to influence policy. Finally, researchers should be involving the wider public: to describe what can be given to society, to demonstrate their value, and also to highlight the waste of human capital and taxpayer money that goes into funding inadequate training.\n\n\nTowards a sustainable workforce\n\nParticipants identified problems with the structure of the workforce in the following key areas (Appendix 2B):\n\nStructure of the system: PIs currently train junior scientists (multiple trainees per PI) in their own image, that is, for a career in academia, though only a small minority will obtain tenure-track faculty positions. Most PIs know little about non-academic careers, even though these comprise the majority of future careers for today’s postdocs. These non-faculty careers are often still looked down upon by those in academia. There is little attention given to training for the careers that the majority of junior scientists will eventually pursue.\n\n“Structure of academic workforce is pyramidal/feudal, generating too many trainees per PI”.\n\nUse of graduate students and postdocs as cheap labor: Junior scientists are primarily treated as cheap labor rather than as participants in a well-rounded training program that prepares participants for a range of clearly identified career options. Postdocs are conflictingly defined as trainees and employees in different situations, which is made possible by the lack of a standardized designation for postdocs and of a clear definition of their duties and responsibilities. There is also no oversight over the number of graduate students and postdocs and whether that number is appropriate given the perceived job market demand. Additionally, there was consensus that funding postdocs through research grants puts them in a vulnerable position and encourages low postdoc salaries allowing for the use of funds elsewhere.\n\n“Postdocs are really hired to produce results, not scientists”.\n\n“Postdoc pay is low so PIs can hire more postdocs to generate more results”.\n\n“Lack of oversight for equal pay for trainees and to prevent exploitation”.\n\nLack of transparency: Problems with workforce sustainability are perpetuated by a lack of information and awareness about the situation, particularly amongst incoming graduate students who seek the increasingly rare academic careers that are still treated as the default career choice by many graduate programs.\n\n“Complete lack of information on number of postdocs”.\n\nFunding and evaluation metrics: Current metrics of evaluation, which are based on the number and impact factor of publications, have resulted in a culture of hyper-competitiveness which discourages creativity, co-operation, risk-taking and original thinking.\n\n“Risk taking not rewarded – No reward for leadership”.\n\nLack of public awareness: Participants also felt a pressing need to make the general public aware of what a scientist really is and what she does, and to more effectively communicate the value of science to the US economy and to humanity as a whole.\n\n“Lack of awareness about how the system operates and functions”\n\nIndividual graduate students and postdocs\n\nEach postdoctoral position should have a defined purpose, including a plan for enhancing the professional skills required in that postdoc’s chosen career path.\n\nGraduate students and postdocs should be proactive about getting career information and carrying out self-evaluation, and discussing these with their mentors. They could also assemble their own career development committee, made up of mentors from various careers of interest.\n\nGraduate student and postdoc associations should collaborate within and between institutions to provide career information and training.\n\nPIs and research groups\n\nPIs should be educated about career paths and trends in the biomedical workforce and how to effectively mentor students and postdocs for available jobs.\n\nPIs should be positively evaluated for diversity of successful career paths taken by their trainees, and not just on the number of trainees that they have placed in research-track careers.\n\nInstitutions\n\nInstitutions should be transparent about the number and funding source of graduate students and postdocs.\n\nAdmission of graduate students could take into consideration their career path and the objective of their training.\n\nIncoming graduate students should be educated about career options and provided with career development advisors.\n\nInstitutions should offer career development courses in all areas of the National Postdoctoral Association core competencies (The National Postdoctoral Association Core Competencies Committee, n.d.).\n\nTrainees should be encouraged to undertake internships outside the lab to gain experience in other career options.\n\nPermanent staff scientist positions should be created with funding structures that remove the competition between the staff scientist and cheaper postdocs or graduate students.\n\nScientists’ involvement in outreach, politics, and entrepreneurship should be encouraged.\n\nFunding agencies and the scientific community\n\nThere should be a standardized designation for all postdocs, irrespective of funding source.\n\nThe purpose and responsibilities of postdocs should be clearly defined.\n\nCaps should be placed on the number of junior scientists per PI.\n\nAll postdocs should receive at least the NIH minimum salary, with a geographical cost-of-living adjustment (US Office of Personnel Management, n.d.), and certain basic benefits.\n\nFunding for postdocs should not be tied to PI research grants.\n\nThe hyper-competitive publish-in-high-impact-journals-or-perish culture should be discouraged and risk-taking, leadership skills and creativity fostered instead.\n\nAs a community, scientists should campaign to educate the public about who scientists are, what they do, and the value of their work.\n\nWithin the academic scientific community, we should foster acceptance of non-academic career path choices.\n\nThere is a clear imbalance between the number of young scientists and the number of jobs available in research. This schism has been widening for the past few decades and producing stress on the scientific workforce which, if unaddressed, will result in a decline in the number of productive young scientists. The fundamental structural flaws in the system need to be addressed; otherwise, as we have seen in the past, simply increasing funding will only postpone and worsen the problem.\n\nYoung scientists need to be engaged in the debate about these changes and advocate for them. They need to come together in collaboration with institutions and the federal government to enforce and implement these changes with a clear discussion of all possible outcomes of these changes.\n\nUltimately the scientific enterprise will grow if the workforce supply and demand are balanced in a sustainable and dynamic fashion, with complete transparency. We can build a highly efficient and productive scientific enterprise if scientists, institutions, governments and industry are all involved and invested in making the necessary changes to the workforce.\n\n\nFunding innovation and training\n\nParticipants identified problems with funding in the following key areas (Appendix 2C):\n\nFunding mechanisms were considered insufficiently diverse: Many participants were in favor of extending the time scales of awarded grants, and cited a need for alternative mechanisms to workhorse grants like the R01, that might permit research projects with alternative aims and organization. In addition, the NIH grant review cycle was seen as inefficiently slow and too bureaucratic to effectively support innovative work. Participants were frustrated at the way that funding agencies were considered to encourage incremental steps in research, thereby discouraging paradigm shifts. They also expressed concern that current funding mechanisms \"kill novel ideas by emphasizing preliminary results“.\n\n“Postdocs should be allowed to apply for grants [directly]”\n\n“Evaluation of grants [is] tied to outdated/improper metrics”\n\nFunding priorities fail to select for long-term productivity: Congressional and institutional trends heavily influence how research money is distributed, such that too much of the available funding is oriented towards ephemerally popular topics, while mature, yet important, research fields are neglected. Concerns were also raised that recent trends in funding favor applied research at the expense of basic research. These priorities undermine the quality and reproducibility of science that is vital to US interests.\n\n“Funding rewards mainly ‘high impact’ publications, [producing] hypercompetitive and dishonest results”.\n\n“Emphasis on translation and the best ‘new’ idea, not reproducibility”\n\nGrant evaluation processes disadvantage young researchers: Institutional leanings in funding agencies were perceived as resulting in funds that are highly centralized; with large grants being awarded to large, well-established labs.\n\n“Bigger names/labs get multiple R01s whereas young/new PIs can’t even get one”.\n\n“Grant success depends maybe too much on previous success; making it much harder for young scientists”\n\nFunding allocation is not subject to post-award review of efficacy: Participants voiced concerns that the current funding paradigm does not lend itself to quantitative, objective analysis of the productivity or quality of research investments. Name recognition and impact factors were reported as weighing too heavily in single-blind study sections, resulting in funds being allocated unscientifically, with few studies of efficacy or predictors of outcome.\n\n“Poorly audited”\n\n“Money spent inefficiently (lack of negotiation, duplication of equipment)”\n\nApproaches to funding were reported as contributing to problems in training and workforce sustainability: Participants noted an insufficient level of direct funding support for postdocs and graduate students, such as through training grants. They also indicated that, by focusing on research productivity alone, funding mechanisms fail to select for graduate and postgraduate education that would aid trainees in developing the skills that would contribute to success in academia or other environments. Funding agencies were also seen as contributing to the negative way that non-academic careers are viewed.\n\n“[The] NIH considers non-academic careers a sign of failure”.\n\n“Students/postdocs used for cheap labor”\n\n“Trainees are often viewed as ‘robots’, leading to burn-out/mental health/work-life balance problems”\n\nGrant application and administration processes are problematic: There was frequent concern regarding the bureaucracy and paperwork involved in applying for and administering grants. Participants characterized the level of effort required to complete auxiliary sections of grant proposals (i.e., outside of specific aims and experimental design) as inefficient, as well as the number of specialized personnel required to submit, review, and administer federal research grants. In addition, several participants found the current peer review system to be insufficiently transparent, and reported that study sections give too little feedback.\n\n“Too much time spent by highest-level scientists writing grants”.\n\nIndividual scientists and research groups\n\nScientists should interact more directly with the public and the government to communicate the benefits of investment in research.\n\nInstitutions\n\nStaff scientists should be supported by grants in order to improve the continuity and accountability of research results within academic labs.\n\nCore facilities should be developed to reduce the resources and specialized expertise required in each lab, allowing smaller lab sizes.\n\nFunding agencies and the scientific community\n\nWe should analyze basic science funding and outcomes to determine how funding award mechanisms affect science.\n\nA greater diversity of funding mechanisms serving smaller labs, younger faculty, and even science enthusiasts within the general public, with an emphasis on encouraging shared, collaborative workspace and core facilities, should be developed.\n\nNew metrics evaluating scientific productivity beyond simple impact factor should be established, along with more post-peer-review and scrutiny of results.\n\nOverall, we would characterize the output of this workshop as a call by young researchers for an increase in the efficiency and reproducibility of science by developing new measures of the quality of research output and of individual researchers’ productivity, and incorporating these criteria into the approval of grants. Participants seemed to agree that this approach, along with some of the other recommendations indicated, would more adequately reflect the priorities of federally-funded science and encourage young researchers to continue careers in basic research.\n\n\nIncentivizing good science\n\nParticipants identified three major classes of behaviors they wished to see in science (in order of popularity, Appendix 2D):\n\nHonesty and integrity: Scientists should pursue the discovery of truth with honesty and integrity, and to the best of their ability; and should continue pushing the boundaries of human knowledge and asking new questions.\n\nCommunication and collaboration: Scientists should share information and ideas freely, both among the scientific community and outside of it. Transparency, openness, sharing, the free exchange of ideas and open dialogue among scientists were all identified as key attributes.\n\nUtility and application of knowledge: Science should produce useful knowledge that can be applied in beneficial ways, with a responsibility to taxpayers to conduct this research with the greatest efficiency possible.\n\nParticipants proposed incentives to encourage the above behaviors:\n\nBetter training in research integrity: Responsible conduct of research education should begin early in graduate school, and ethics discussions should be commonplace.\n\nTracking investments in trainees: Funding agencies should maintain centralized information on trainee outcomes and make these data available to prospective trainees to encourage investment in students’ and fellows’ education.\n\nNew metrics of integrity: While current publication metrics encourage flashy publications, metrics should be created to reward integrity and honesty. These measures could include peer review contributions (whether pre- or post-publication); whether qualitative or quantitative, these could influence grant and job applications.\n\nOpen data and reducing the “minimal publishable unit”: Journals could require data uploads prior to publication and raw data access during revision and/or following publication. This would encourage careful record-keeping and unbiased analysis through the scientific process. Furthermore, many results (especially negative and contradictory results) could be published under new models that do not require the time and resource investment of a traditional paper.\n\nIndividual graduate students and postdocs\n\nGraduate students and postdocs should be able to anonymously provide feedback on their training experiences and outcomes, ideally using the IDP as a framework.\n\nPIs and research groups\n\nOpen data access policies and publication of negative results should be encouraged.\n\nInstitutions\n\nAdequate training on the responsible conduct of research and critical thinking skills should be provided.\n\nAnonymous evaluation of available training by graduate students and trainees should be aggregated at the departmental level and used to form part of a training score for the department and institution.\n\nFunding agencies and the scientific community\n\nMetrics of community-minded behavior (publishing negative results, peer review activity) should be taken into account when awarding grants.\n\nA website should be established to track graduate student and postdoc outcomes across institutions.\n\nA training score for departments and institutions should be considered during grant review.\n\nThe output of this workshop was a call by young researchers for incentivization of transparency and honesty in science, by developing new metrics and possibly incorporating these criteria into funding mechanisms. In particular, we propose the creation of a website for trainees to anonymously publish feedback on their training experiences and outcomes, ideally using the IDP (Fuhrmann et al., n.d.) as a framework. Trainees might complete an IDP, then later return to the site to report on their progress. Data, aggregated at the departmental or program level, would form part of a training score for the department and institution. This would permit prospective students and fellows to factor this information into their career decisions, thereby rewarding institutions that place an emphasis on training with improved student and fellow recruitment. Incorporating this score into the grant review process would encourage departments to invest in training. The website could also facilitate publication of institutions’ training plans that outlines available career development opportunities. This could encourage the creation of de facto universal standards for training.\n\n\nSymposium organization\n\nThe Future of Research Symposium was organized by a group of postdoctoral scholars from universities in the Boston area, including Boston University, Harvard University, Harvard Medical School, Tufts University, Brigham and Women’s Hospital, the Massachusetts Institute of Technology, Brandeis University, and the Dana Farber Cancer Institute. The symposium was hosted at Boston University through a partnership with Boston University’s Graduate Women in Science and Engineering (GWISE).\n\nSpeakers from academia and industry who have led national discussions participated. Henry Bourne opened the symposium with a keynote outlining the changes he thinks must be made to the scientific infrastructure. A panel comprising Sibby Anderson-Thompkins (Director, Office of Postdoctoral Affairs, University of North Carolina at Chapel Hill), Galit Lahav (Associate Professor, Harvard Medical School), Graham Walker (American Cancer Society Professor, HHMI Professor, Massachusetts Institute of Technology), David Glass (Executive Director, Novartis Institutes for Biomedical Research), and Richard Roberts (Chief Scientific Officer, New England Biolabs) summarized weaknesses and potential improvements in the current training system. A second panel comprising Marc Kirschner (John Franklin Enders University Professor of Systems Biology, Harvard Medical School), Michael Teitelbaum (Senior Research Associate, Harvard Law School), Naomi Rosenberg (Dean of the Sackler School of Graduate Biomedical Sciences, Tufts University), and Cynthia Furhmann (Dean of Career & Professional Development in the Graduate School of Biomedical Sciences, University of Massachusetts Medical School) discussed issues pertaining to the scientific workforce and their implications for the future of science in the United States.\n\nAt the end of each workshop, participants were asked to fill out a short exit survey (full text in Appendix 3; individual comments from each workshop in Appendices 3A–D). The survey was designed to address three objectives. First, in order to assess how well the workshop format was working and how it could be improved, participants were asked to rate on a five-point scale how well the session addressed the stated objective, and how well they were able to arrive at meaningful solutions.\n\nThe second objective of the exit survey was to determine whether or not participants felt they had reached a consensus during the workshop, and to gauge the importance participants placed on reaching consensus about these issues. As the primary official report arising from a symposium intended to give voice to early-career scientists, this document should reflect the views expressed at the conference. However, it was unclear whether that aim would be best accomplished by representing all or most of the opinions expressed during the workshops, or by presenting only those ideas about which participants had reached consensus. Participants were therefore asked to rate on a five-point scale whether or not their group reached a consensus and how important they thought it was to have either a consensus or a diversity of views regarding these issues.\n\nFinally, we asked participants to list any specific suggestions they might have about next steps to be taken after the symposium, such as ideas about people or organizations we should contact. The results of the survey are summarized in Figure 2. The survey indicated that participants cared both about reaching a consensus and having a diversity of ideas. Participants also indicated that the workshops were more successful in generating multiple solutions than in finding unanimity.\n\nWhile we did not strictly monitor the attendance at the symposium, registration data suggested that the majority of participants were postdocs and graduate students. Of 658 registrants, 344 were postdocs, 140 were graduate students, and the remainder included a mix of professors, instructors, journalists, administrators, research technicians, and research scientists from both academia and industry.\n\nFor detailed information on the requirements for preparing a symposium please see: The Logistics of Organizing the Future of Research Symposium (Mazzilli et al., 2014).\n\n\nMedia response and online discussion\n\nThe symposium received a wide variety of feedback and responses during and after the event, from both social media and the press, which continues to foster discussion.\n\nDuring the symposium we engaged a large number of participants, both local and remote, using the #FORsymp hashtag on twitter, and the @FORsymp twitter account, to leave comments and ask questions. Figure 3 shows examples of significant tweets and questions received during the symposium. A collection of salient moments from the symposium was also gathered by Alberto Roca (@minoritypostdoc) into a Storify (Roca, 2014).\n\nComments were also received from interested non-local parties and the symposium was particularly well tracked in the UK and Australia, as illustrated in Figure 4. The comments reinforced that these problems are not unique to Boston or the US; this is part of the discussion we hoped to generate further afield.\n\nImmediately after the symposium, significant attention was focused on the Future of Research in the publication of an article by Carolyn Johnson in the Boston Globe, “Glut of postdoc researchers stirs quiet crisis in science” (Johnson, 2014). The article remained the most viewed on the Boston Globe website for several days thereafter and generated a wide range of responses, including comments on the article itself:\n\n“Not only is it unconscionable that these highly educated scientists - after being encouraged to pursue PhD’s by universities that know what career path difficulties lie ahead including years of being paid paltry annual salaries starting as low as $42,000, but it also makes being able to live a decent lifestyle and repaying their student debt impossible; now unforgivable even by bankruptcy”.\n\n“Boom-and-bust syndromes have plagued science and engineering markets for at least fifty years. Over and over, we’ve heard laments like those in the current article. Yet little, if anything, seems to have changed for the better. The problems, and the lack of genuine progress, have been vexing”.\n\n“One possible partial solution would be to treat academic research groups as long-term productive teams. Pay post-docs what they’re worth, and make their positions permanent mid-level research positions. There’s plenty of people in the pipeline who love doing science who don’t really want to be the head of their own lab, but we’re trained as though that’s the only option”.\n\nThe Boston Globe itself compiled a Storify (The Boston Globe, 2014) on responses to the article from social media, particularly through use of the hashtag #lifeafterPhD, and published additional opinion pieces, including “Let’s change the system for postdocs” and “Postdocs in limbo? Expand your options” (Fuentes-Medel, 2014; Kirshenbaum, 2014).\n\nThe article also generated further discussion on reddit:\n\n“I think it is fairly absurd that the NIH estimate for number of postdocs working in the US has an error of ±15,500 people”.\n\n“Is it far-fetched to think that academia is headed toward a tipping point? Under the current situation, nobody currently in grad school or academia more broadly is going to recommend STEM as a career path to anyone, in particular their children”.\n\n“It’s frustrating because being a scientist is advertised as a stable job to people at every level of the education system until you’re actually in grad school”.\n\nThere was also significant commentary on Slashdot:\n\n“If there was a genuine shortage [in STEM], you’d see sharp increases in salary levels. There’s just a shortage of qualified people willing to work for much less than they’re worth”.\n\n“Historically university posts were open to people with a BA (e.g. John Wesley and John Newman at Oxford in the 18th and 19th century). That it now takes a PhD and post doctoral work to get the same post means that we are training too many. Therefore the only solution is to row back on the PhDs being generated; given that governments are looking for money saving measures, this would seem an obvious starting point”.\n\n“It’s crazy that we have these vast hordes of people trained up and desperate to work hard for scientific progress. But our economy can’t find a way to provide them with jobs doing science”.\n\nConversations on the LinkedIn group “PhD Careers Outside Academia”, “The Postdoc Holding Tank” on October 5th and “What we already know…” on October 9th reiterated that this is not a new problem; the comments highlighted the dismal outlook among members of the community stemming from the lack of progress over the last decade. However, it is this very lack of change and the wider appreciation of the problem outside the academic community that we wish to target with the symposium and the publication of this document.\n\nIn the aftermath of the symposium, significant attention has been drawn to the event in academic media: in “Postdocs Speak Up” (Benderly, 2014), Science Careers magazine discussed the role of postdocs in advocating for themselves; an interview was featured in the scientific podcast Beta Sandwich; and the National Postdoctoral Association published an article about the symposium in its publication, the POSTDOCket.\n\nWe intend to continue bringing this conversation into the graduate student and postdoc community. Already, a poster (McDowell et al., 2014b) has been presented at the Out to Innovate 2014 Conference held by the National Organization of Gay and Lesbian Scientists and Technical Professionals, Inc. (NOGLSTP) and Out in Science, Technology, Engineering, and Mathematics, Inc. (oSTEM) at Georgia Tech (Atlanta, GA, November 8–9, 2014). The poster, which won the Leadership Poster Prize, generated a great deal of discussion regarding the intersection between postdoc and graduate student issues and issues facing underrepresented minorities.\n\n\nConclusion\n\nThe workshops represented an opportunity for junior scientists to come together and discuss problems with the current scientific enterprise, and they produced an abundance of suggested solutions. Given the limited time of the workshops and the varied background of the participants in terms of their perspective on the current system and its challenges, a consensus on specific steps to be taken was not achieved. There were, however, certain common themes that require further discussion; because of the interconnected nature of these issues, affecting change will require a deeper understanding of both the causes of the problems and the effects of the proposed solutions. As a starting point for a larger and longer discussion, we have distilled three main proposals from the workshops and continued discussions among the organizers that can be implemented at all levels, from individual postdocs to institutions such as the NIH.\n\nFirst, we recommend increased connectivity among junior scientists as well as between junior scientists and other segments of the scientific community. Postdocs and graduate students frequently conduct their research in isolation, as their work is rewarded primarily upon the basis of its novelty and independence, and as they are all competitors for a vanishingly small pool of advanced academic positions. The sense of isolation is particularly strong among postdocs, as many uproot themselves from their professional networks to take positions in geographically distant institutions without an accompanying cohort (such as in graduate school). This isolation precludes awareness of larger institutional issues and makes it more difficult for postdocs to advocate for themselves and bring about positive change. Postdocs and graduate students also must connect with other stakeholders in science so as to participate in the ongoing discussions about changes in training, funding, and other important policy issues. Finally, postdocs and graduate students should come together to define their position as major stakeholders in the research enterprise. While as individuals, junior scientists are temporary, replaceable, and largely anonymous, together they constitute the engine of the academic workforce. As such, they need to take collective action to ensure that their interests are protected as they work to maximize scientific output and efficiency (Cain et al., 2014). Only by bringing all stakeholders together will science be able to effectively grow and adapt to current and future challenges.\n\nSecond, we recommend increased transparency in trainee numbers and outcomes. Currently, national conversations decrying the “STEM shortage”, as well as a lack of accessible information about the state of the workforce, create skewed perceptions regarding the demand for PhDs among many beginning biomedical graduate students. Students may become aware of the pyramidal structure of the academic workforce only late in their training. To remedy this, the number of graduate students and postdocs at all institutions should be made publicly available, together with information on career outcomes. Collecting and publishing information on career outcomes should be made a condition of an institution receiving NIH funding. Many institutions already collect this information at regular intervals, but lack a centralizing node to distribute it, and to compare the effect of their leadership. These organizations have a moral imperative to share this information; its dissemination will enable informed career and policy decisions. In addition, former students and postdocs should have a forum in which to anonymously report the outcomes of their training and subsequent career moves. Furthermore, there is a significant need to better define the role and purpose of the postdoc position. We advocate for transparency in terms of defining expectations of the balance between employment and training in individual postdoc appointments.\n\nFinally, we call for increased investment in postdocs through financial independence from PI research grants and increased accountability for the quality of postdoc training. Currently, many postdocs have little power to freely pursue creative research directions and individual professional development plans, or to negotiate for necessary employment benefits. We propose two possible mechanisms for increasing postdoc autonomy. First, postdocs should not be supported by research grants, but rather exclusively by individual training fellowships. With this increased intellectual independence, postdocs would be allowed to pursue projects of mutual interest to themselves and their mentors. This creates a much-needed line between staff scientists and technicians, who may be paid and directed by research grants, and postdoctoral scholars, who should be focused on training and development. Second, the institutions employing, and the agencies funding, postdocs should seek increased accountability for their training through direct postdoc feedback to the funding agency. These reports of training experience and support given by PIs, departments, and institutions should be used in evaluating grants for award and renewal. Furthermore, some of this information, properly anonymized and aggregated, could be used to create a publicly accessible “training score” for departments; this metric would incentivize excellence in mentoring to maintain competitiveness in recruitment of young, talented scientists.\n\nAs the source of future scientific leadership, postdocs and graduate students are uniquely placed to influence the direction and culture of the research enterprise. To be most effective, however, we must educate ourselves about the prevailing conditions affecting the workforce and sustainability of research, and their historical and institutional bases. The voices of junior researchers must command a greater audience in the present discussion; additionally, as we take our places as the next generation of independent academic scientists, we can influence the culture, efficiency, and integrity of research from within. From both the attendance at the symposium and the ongoing coverage of the event and issues discussed, it is clear that junior scientists are invested in and passionate about these issues. We all must now rise to the challenge of taking action to build a sustainable, productive, and equitable scientific community.\n\n\nData availability\n\nF1000Research: Dataset 1. Dataset of Future of Research Symposium, 10.5256/f1000research.5878.d39822 (McDowell et al., 2014d)", "appendix": "Author contributions\n\n\n\nAll authors were involved in the preparation and revision of the manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declare that no grants were involved in supporting this work. The authors wish to thank our generous sponsors without whom the symposium would not have been possible: Novartis Institutes for Biomedical Research, the National Academy of Sciences, Harvard Medical School, the American Society for Cell Biology, the Harvard Medical School Postdoc Office, the Harvard Medical School Department of Systems Biology, the Harvard Medical School Department of Genetics, Monsanto Company Inc., Addgene, the Boston University Office of Professional Development and Postdoctoral Affairs, New England BioLabs Inc., the Tufts University Postdoctoral Office, Miltenyi Biotech, and Nature Jobs.\n\n\nAcknowledgements\n\nWe are grateful for the time, counsel, and support of many advisors without whom the event would not have been possible. We are especially indebted to Becky Ward for inspiration and advice at every step of the way. We also thank Manu Sarna for teaching us how to moderate workshops, Henry Sauermann for advising us on surveys, Judy Glaven for feedback and perspective, Michelle Brook for guidance with blogging and social media, and David Cameron for assistance with promoting and framing the event. We are also grateful to Rosy Haskins, David Riglar, Dmitry Schvartsman and Ferdinando Pucci for their helpful comments on the manuscript.\n\nWe thank GWISE (Graduate Women in Science and Engineering) for partnering with us to host the event at Boston University (BU), Linda Hyman and the BU postdoc office, and the many BU administrators in financial offices that have and continue to support the symposium with their efforts.\n\n\nReferences\n\nAlberts B: Overbuilding research capacity. Science. 2010; 329(5997): 1257. PubMed Abstract \| Publisher Full Text\n\nAlberts B, Kirschner MW, Tilghman S, et al.: Rescuing US biomedical research from its systemic flaws. 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Publisher Full Text\n\nStephan P: How Economics Shapes Science. Cambridge, MA: Harvard University Press. 2012a. Reference Source\n\nStephan P: Research efficiency: Perverse incentives. Nature. 2012b; 484(7392): 29–31. PubMed Abstract \| Publisher Full Text\n\nStrategic Evaluations, Inc. Training in Education and Critical Research Skills Program (TEACRS), Tufts University School of Medicine. A Comparison of Active Trainees’ Research Progress. Durham, NC. 2014.\n\nTeitelbaum MS: Research funding. Structural Disequilibria in Biomedical Research. Science. 2008; 321(5889): 644–645. PubMed Abstract \| Publisher Full Text\n\nThe Boston Globe: Research community responds to “Glut of postdoc researchers stirs quiet crisis in science” by Carolyn Johnson. 2014. Reference Source\n\nThe National Postdoctoral Association Core Competencies Committee: (n.d.). The NPA Postdoctoral Core Competencies. National Postdoctoral Association. 2007–2009. Reference Source\n\nUS. Office of Personnel Management. 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Reference Source" }	[ { "id": "6854", "date": "09 Dec 2014", "name": "Paula E Stephan", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe report provides an excellent portrayal of conditions faced by postdoctoral researchers and graduate students in the biomedical sciences in the United States and it makes a number of constructive suggestions for reforms that could make the system more “early career” friendly. For example, the authors advocate increased transparency so that doctoral students and postdoctoral scholars will know the career outcomes of those who proceeded them and a change in the ratio of individuals who staff labs between graduate students, postdocs, technicians and staff scientists. They also advocate a greater focus on facilitating the learning of soft skills while in training and a redirection of resources towards younger researchers and away from extremely senior researchers. (Over 7% of all NIH R01 supported PIs are currently 66 or older and about 3% are 36 or younger.) I would add to this list an increase in the salary of postdoctoral trainees: the current system, with its low pay for postdoctoral researchers, actively encourages an over reliance on postdoctoral trainees in the lab. I would also shift support of graduate students to training grants and away from graduate research assistantships. This would allow more control over the quality of training and the number of individuals trained. I would also note, as someone who has studied the funding of science and the biomedical work force for many years, that Vannevar Bush’s vision was to support graduate students and postdoctoral researchers on fellowships, not on graduate research assistantships. His vision was to train future researchers, not to provide support for trainees to staff labs during their years of training. It was only in the late 1950s and early 1960s that the system began to increasingly tilt towards supporting students as graduate research assistants. It is also interesting to remember that “over training” in the biomedical sciences has been a major issue for at least 40 years. As early as 1976, an NRC report concluded that a “slower rate of growth in the labor force in these fields [the biomedical sciences] was advisable.” And in 1998, the NRC report, “Trends in the Early Careers of Life Scientists,” chaired by Shirley Tilghman, called for restraint in the number of PhDs produced and increased use of training grants relative to graduate research assistantships.", "responses": [] }, { "id": "6856", "date": "11 Dec 2014", "name": "Kenneth D. Gibbs Jr.", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe report by McDonnell et al. aims to provide a summary of the Future of Research Postdoctoral Symposium that took place in October 2014. The work of this group is commendable, and has the potential to inform on-going policy debates about how to improve the research enterprise. The article has some interesting findings and recommendations. However, I feel there are two important issues that prevented me from accepting without reservation: focus and tone.1. Focus. I found the manuscript, as written, very difficult to follow. My sense is that in an effort to be comprehensive, the focus became lost (as one who often writes long and unwieldy drafts, I have much sympathy). In my view, readability of the paper would be enhanced by organizing it in three sections:Context/Background. Here the authors can use data and other policy reports to describe some of the structural changes that have occurred in biomedicine in the past few years (e.g. stalled funding, growing number of trainees, etc.). They can then couple these changes with some of the issues that have resulted from this (e.g. an increased sense of competition to a level where it’s no longer helpful; the favoring of incremental science over exploration, etc.). From this point, they can say the purpose of this symposium: adding the voices of this groups of postdocs to the debate. Symposium. Clearly and concisely describe the symposium. List the four or five major goals/foci of the symposium (likely the workgroups). Then, for each, use a table to describe: 1. The central issue, 2. How postdocs describe the problem (only list the main points, and if possible include the percentages of postdocs conceptualizing the problem in that manner), and 3. The proposed solutions (with individual students, PIs/research groups, institutions, and funding agencies as their own column). Four charts would significantly enhance readability. All additional information can be put in the supplement. Summary recommendations. This could include a very brief nod to the media coverage (most of that should be in the supplement), the recommendations (currently the conclusions), and future recommendations.2. Tone. The paper read as a policy report, editorial, and meeting minutes all as one. There are many points where the authors make declarative statements but don’t offer any citations. Two examples of many include:“Specifically, the hyper-competition that we have all experienced, which stunts scientific curiosity and productivity, breeds fabrication and carelessness in the publication of data, and leads to a waste of valuable resources and intellectual capital, must be alleviated.” It is not clear that all have experienced this and would make these conclusions.The paragraph starting with this sentence: “In spite of the number of years spent in pre- and postdoctoral training, only a handful of scientists feel that they are adequately pre- pared for any job other than conducting research.” While career preparation is highly variable, it is an overstatement to say \"only a handful feel prepared\" for careers outside of research. The authors would be well served by making it clear when they are stating something that is a fact, versus when they are conveying the opinions of the participants. Additionally, sometimes the term “we” is used, and it’s unclear who it is referring to—the paper’s authors, FOR Symposium attendees, the broader research community. Please be clear to whom you are referring. Finally, the manuscript has something that seemed to be a major contradiction. In the executive summary, the authors say their report “represents a united voice of young biomedical scientists, conveying our concerns about the sustainability of the research enterprise and our hopes for change.” However, on at least two occasions, they describe the challenge in reaching consensus among FOR Symposium participants“Overall, the respondents’ concerns and criticisms centered on a few key themes; however, there was disagreement regarding which issues are most important to the future of groundbreaking and sustainable science.” “Participants also indicated that the workshops were more successful in generating multiple solutions than in finding unanimity.”Even if there is not a “uniform voice” the symposium findings are still important. Don’t feel the need to overstate conclusions.Again, I feel the work is important. I fully expect the authors will be able to address my comments, at which point I would enthusiastically approve it.", "responses": [] } ]	1	https://f1000research.com/articles/3-291
https://f1000research.com/articles/3-305/v1	12 Dec 14	{ "type": "Research Note", "title": "Binding of a fluorescence reporter and a ligand to an odorant-binding protein of the yellow fever mosquito, Aedes aegypti", "authors": [ "Gabriel M. Leal", "Walter S. Leal", "Gabriel M. Leal" ], "abstract": "Odorant-binding proteins (OBPs), also named pheromone-binding proteins when the odorant is a pheromone, are essential for insect olfaction. They solubilize odorants that reach the port of entry of the olfactory system, the pore tubules in antennae and other olfactory appendages. Then, OBPs transport these hydrophobic compounds through an aqueous sensillar lymph to receptors embedded on dendritic membranes of olfactory receptor neurons. Structures of OBPs from mosquito species have shed new light on the mechanism of transport, although there is considerable debate on how they deliver odorant to receptors. An OBP from the southern house mosquito, Culex quinquefasciatus, binds the hydrophobic moiety of a mosquito oviposition pheromone (MOP) on the edge of its binding cavity. Likewise, it has been demonstrated that the orthologous protein from the malaria mosquito binds the insect repellent DEET on a similar edge of its binding pocket. A high school research project was aimed at testing whether the orthologous protein from the yellow fever mosquito, AaegOBP1, binds DEET and other insect repellents, and MOP was used as a positive control. Binding assays using the fluorescence reporter N-phenyl-1-naphtylamine (NPN) were inconclusive. However, titration of NPN fluorescence emission in AaegOBP1 solution with MOP led to unexpected and intriguing results. Quenching was observed in the initial phase of titration, but addition of higher doses of MOP led to a stepwise increase in fluorescence emission coupled with a blue shift, which can be explained at least in part by formation of MOP micelles to house stray NPN molecules.", "keywords": [ "Over the past decade progress towards our understanding of the molecular basis of mosquito olfaction has been remarkable. It was not until the sunset of last century that odorant receptor (OR) genes have been identified in the genome of the fruit fly", "Drosophila melanogaster1–3 and thereafter in mosquitoes and various insect species (see review4)", "and less than a decade since the unique topology of ORs", "with an intracellular N-terminus and an extracellular C-terminus5", "has been elucidated. Although previously known from moth species6", "it was about a decade ago that the first odorant-binding proteins (OBPs) from mosquitoes have been isolated and identified7. By now the complete repertoire of olfactory genes", "including OBP", "OR and ionotropic receptor (IR) genes", "have been identified in the three major mosquito species: the yellow fever mosquito", "Aedes aegypti8", "the malaria mosquito", "Anopheles gambiae9", "and the southern house mosquito", "Culex quinquefasciatus10. There is growing evidence in the literature that OBPs and ORs play a crucial role in the sensitivity and selectivity of the insect’s olfactory system4. Mosquito ORs have been deorphanized and demonstrated to be essential for the reception of physiologically and behaviorally relevant odorants9", "11", "including oviposition attractants12–14", "insect repellents15 and a signature compound (sulcatone) for human host preference16. Elucidation of the three-dimensional (3D) structures of mosquito OBPs17–21 along with knockdown experiments22", "23 and binding assays24–27 strongly suggest that these olfactory proteins are involved in the transport of odorant from the ports of entry of olfactory sensilla (the pore tubules) to ORs housed on dendritic membranes of olfactory receptor neurons." ], "content": "Introduction\n\nOver the past decade progress towards our understanding of the molecular basis of mosquito olfaction has been remarkable. It was not until the sunset of last century that odorant receptor (OR) genes have been identified in the genome of the fruit fly, Drosophila melanogaster1–3 and thereafter in mosquitoes and various insect species (see review4), and less than a decade since the unique topology of ORs, with an intracellular N-terminus and an extracellular C-terminus5, has been elucidated. Although previously known from moth species6, it was about a decade ago that the first odorant-binding proteins (OBPs) from mosquitoes have been isolated and identified7. By now the complete repertoire of olfactory genes, including OBP, OR and ionotropic receptor (IR) genes, have been identified in the three major mosquito species: the yellow fever mosquito, Aedes aegypti8, the malaria mosquito, Anopheles gambiae9, and the southern house mosquito, Culex quinquefasciatus10. There is growing evidence in the literature that OBPs and ORs play a crucial role in the sensitivity and selectivity of the insect’s olfactory system4. Mosquito ORs have been deorphanized and demonstrated to be essential for the reception of physiologically and behaviorally relevant odorants9,11, including oviposition attractants12–14, insect repellents15 and a signature compound (sulcatone) for human host preference16. Elucidation of the three-dimensional (3D) structures of mosquito OBPs17–21 along with knockdown experiments22,23 and binding assays24–27 strongly suggest that these olfactory proteins are involved in the transport of odorant from the ports of entry of olfactory sensilla (the pore tubules) to ORs housed on dendritic membranes of olfactory receptor neurons.\n\nThere are typically two binding assays to “de-orphanize” OBPs, i.e., to measure their binding affinities and specificity towards physiologically and behaviorally relevant odorants (ligands). They are the cold binding assay28 so named because – as opposed to its predecessors - it does not require radioactive ligands and a fluorescence reporter assay29,30. The former is based on separation of bound and unbound OBPs, followed by extraction of bound ligands and their quantification by gas chromatography. In the latter a test OBP is bound to a fluorescence reporter, N-phenyl-1-naphthylamine (NPN, Figure 1), and subsequently increasing amounts of a test ligand are added. Decreasing NPN fluorescence emission is inferred as NPN displacement, i.e., the test ligand is assumed to compete for the binding site initially occupied by NPN. The fluorescence reporter assay is such a facile method that we envisioned it could be used even in a high school research project.\n\nN-phenyl-1-naphthylamine (NPN) is widely used in binding assays with insect OBPs. (5R,6S)-6-acetoxy-5-hexadecanolide (MOP) is an attractant first isolated from eggs of Cx. quinquefasciatus37, but it is known to bind not only to CquiOBP1, but also to its orthologous proteins, i.e., AaegOBP1 and AgamOBP119.\n\nThe 3D structures of the malaria mosquito OBP, AgamOBP121 bound to polyethylene glycol (PEG) and AgamOBP1 complex with DEET18, suggested that AgamOBP1 could be a DEET carrier. For this high school project we asked the question whether DEET and other insect repellents (picaridin, IR3535, and PMD) would bind to AaegOBP131 (also named AaegOBP3932,33), an orthologue of AgamOBP1 from the yellow fever mosquito with similar 3D structure20. In the course of this investigation, we found evidence suggesting that AaegOBP1 might bind simultaneously the fluorescence reporter and an odorant.\n\n\nMaterials and methods\n\nAaegOBP1 (AY189223)31 was expressed in LB medium with transformed BL21(DE3) cell (Agilent Technologies, Santa Clara, CA) according to a protocol for periplasmic expression of insect OBPs34. Proteins were extracted with 10 mM Tris-HCl, pH8 by three cycles of freeze and thaw35. After centrifuging at 16,000×g to remove debris, AaegOBP1 was isolated from the supernatant and purified by a series of ion-exchange and gel filtration chromatographic steps, as previously described20. The purest fractions were combined and desalted, according to a previous protocol20. Then, AaegOBP1 was delipidated following an earlier protocol36 with small modifications. In short, hydroxyalkoxypropyl-dextran Type VI resin (H2658, Sigma, St. Louis, MI) (1g) was suspended in HPLC grade methanol (20 ml), transferred to a glass column (i.d., 8.5 mm) with a stopper, washed with 60 ml of methanol and then washed and finally equilibrated with 50 mM citric acid buffer, pH 4.5. AaegOBP1 (ca. 2 mg per batch) in 50 mM citric acid buffer, pH 4.5 was mixed with the equilibrated resin in a 15 ml Falcon tube, and incubated at room temperature in a high speed rotating extractor (Taitec, Tokyo, Japan) at 50 rpm. The mixture was then transferred to a glass column and AaegOBP1 was eluted with citric acid buffer and analyzed by SDS-gel electrophoresis. The purest fractions were desalted on four 5-ml HiTrap desalting columns (GE Healthcare Life Sciences) in tandem by using water as mobile phase. Protein concentration was measured by the Quick Start Bradford Protein Assay (Bio-Rad, Hercules, CA).\n\nFluorescence measurements were done on a RF-5301 spectrofluorophotometer (Shimadzu, Kyoto, Japan) equipped with a magnetic stir bar. Samples in a 2-ml cell were excited at 337 nm, with the emission spectra recorded from 350 to 500 nm. Both emission and excitation slit were set a 5 nm. Data were recorded in high sensitivity, with automatic response time, fast scan speed, and sample pitch of 1 nm. AegOBP1 samples (10 µg/ml; ca. 0.7 µM, unless otherwise specified) were prepared in 100 mM ammonium acetate buffers. NPN titration were performed with acetate buffers pH 5.5 or pH 7. The other experiments, unless otherwise indicated, were done with acetate buffer pH 7. The fluorescence reporter and ligands were added by 0.5 or 1 µl aliquots of 1, 5, or 10 mM solutions in methanol. For displacement assays, 1 µl of 10 mM NPN (unless otherwise specified) was added, the solution was stirred in the cell for at least 10 min, stirring was ceased and spectra recorded. Then one aliquot of the test ligand was added, mixed for 2 min, and then the spectra were recorded. For NPN titration, the protein sample was stirred for 2 min, spectra recorded, 0.5 or 1 µl of 1 mM NPN solution was added and stirred for 2 min before recording. To avoid possible interferences, the light path was open only during recording and stirring was ceased at least 10 s before spectra were acquired.\n\nData were analyzed with GraphPad Prism 6 (La Jolla, CA). For clarity, traces were reconstructed with GraphPad by transferring recorded data without normalization. To draw Figure 4, data were normalized (fluorescence recorded with AaegOBP1 and NPN, 100%) and for each concentration of the ligand mean ± SEM from three experiments were calculated in an Excel datasheet and transferred into Prism. Dissociation constants for NPN were determined by nonlinear regression curve fitting, one site and specific binding. MOP dissociation constant was calculated by measuring its competition for NPN binding. Thus, data were analyzed by nonlinear regression curve fitting (one site fits Ki), using the concentration of NPN (typically 5000 nM as HotNM) and Kd for NPN in nM (HotKdNM).\n\nNPN and DEET (N,N-diethyl-3-methylbenzamide) were acquired from Sigma-Aldrich. MOP and PMD (p-mentan-3,8-diol) were gifts from Bedoukian Research, Inc. Picaridin (butan-2-yl 2-(2-hydroxyethyl)piperidine-1-carboxylate) and IR3535 (ethyl 3-[acetyl(butyl)amino]propanoate) were gifts from Dr. Kamal Chauhan (USA, ARS, Beltsville).\n\n\nResults and discussion\n\nIn preparation for binding assays of AaegOBP1 with insect repellents, we first measured the dissociation constant, Kd, for NPN: 3.31 ± 0.48 μM (n = 3). Subsequently, we measured fluorescence quenching by adding aliquots of insect repellents to a protein solution pre-equilibrated with 5 μM of NPN. To minimize solvent effect and reduce experimental error, we added 0.5 μl of 5 mM solutions of test ligands using a 2 μl pipette. As a positive control, we used a racemic solution of the mosquito oviposition pheromone (5R,6S)-6-acetoxy-5-hexadecanolide (MOP)37 (Figure 1), which has been previously demonstrated with the cold binding assay to bind to AaegOBP1 with apparently high affinity19. Titration with DEET showed minor reduction in fluorescence intensity (Figure 2) thus suggesting weak binding. By contrast, addition of 1.25 μM MOP led to almost one-third reduction in fluorescence intensity. Titration with other commercially available insect repellents, namely, picaridin, IR3535, and PMD gave similar results as DEET. Although our results suggest that all four repellents bound to AaegOBP1, it seems their affinities were too low to accurately measure dissociation constants. To complete the project and allow the high school investigator to measure at least one dissociation constant, we titrated MOP and this experiment led to unexpected and interesting results.\n\nNPN bound to AaegOBP1 was excited at 337 nm and its emission spectra (black trace) was recorded. Then, increasing doses of DEET were added and finally one aliquot of MOP was added.\n\nAddition of MOP to solutions of AaegOBP1 pre-incubated with NPN caused a stepwise decrease in fluorescence intensity (2.5 μM to 10–12.5 μM doses), but rather than saturation further addition of MOP led to fluorescence increase and a blue shift. The senior investigator assumed it was an experimental error and repeated the experiments (Figure 3). Quenching was observed when MOP was added up to 10–12.5 μM, but fluorescence increased thereafter and the maxima excitation wavelength shifted: AaegOBP1-NPN only, max 445 nm; AaegOBP1-NPN plus 2.5 μM MOP, 449 nm; AaegOBP1-NPN plus 20 μM MOP, 433 nm. Although unlikely, we tested whether this unexpected fluorescence emission could be generated by MOP itself when bound to AaegOBP1. The fluorescence emission levels generated even with AaegOBP1 plus 20 μM MOP (highest dose and no NPN) were indeed too low (Figure 3) to explain the overall increase in fluorescence. We repeated these experiments and observed a clear U-shape curve with a minimum at 10–12.5 μM (Figure 4). We measured the dissociation constant for MOP (2.64 ± 0.16 µM, n = 3) by considering only the first phase of the curve, i.e., by using the data generated by quenching or NPN replacement. Although the above experiments were conducted with reasonable low concentrations of ligands as compared to typical experiments29,30, we next examined the possibility of micelle formation with higher doses of MOP. We repeated titration of MOP using the same doses of the ligand, but reducing the concentrations of protein (0.35 µM) and fluorescence reporter (NPN, 2.5 µM) (Figure 5). When added to ammonium acetate buffer at pH 7 (Figure 5B) or AaegOBP1 in the same buffer (Figure 5A), NPN fluoresced with emission maxima at 469 and 446 nm, respectively. Addition of MOP (2.5–10 µM) led to quenching of NPN in protein solution, but no significant change of NPN fluorescence in buffer solution. Addition of higher doses of MOP to a buffer solution, however, suggested the formation of micelles given the increase in fluorescence and blue shift observed at 12.5 and 15 µM of MOP (Figure 5B), although we do not know the critical micelle concentration for MOP. The increase in fluorescence and blue shift were more pronounced in the presence of protein (Figure 5A). It is, therefore, possible that the increase in fluorescence is a combination of micelle formation and other factor(s), which cannot be dissected by these experiments.\n\nFollowing addition of NPN, fluorescence emission spectra were recorded with increasing doses of MOP. Note the decrease in fluorescence intensity (quenching) as the doses increases up to 10 µM and an increase in fluorescence and blue shift at higher doses. In a separate experiment, included in the lower part of the figure for comparison, fluorescence emission spectra were recorded with AgamOBP1 alone and after addition of MOP, but in the absence of NPN.\n\nEmission maxima were normalized to display mean ± SEM from three experiments. MOP dissociation constant was calculate for the decreasing phase (0–12.5 µM). Note the increase in fluorescence emission thereafter.\n\n(A) NPN (2.5 µM) was added to a solution of AaegOBP1 (0.35 µM) in ammonium acetate buffer, pH 7. (B) NPN (2.5 µM) was added to ammonium acetate buffer, pH 7. In both cases, increasing aliquots of MOP were added and emission spectra were recorded.\n\nLastly, we compared the fluorescence emission spectra obtained by titrating AaegOBP1 solutions at low and high pH values (Figure 6). Interestingly, NPN showed a higher affinity for AaegOBP1 at pH 5.5 than at pH 7. Additionally, the emission spectra at low pH were blue shifted relative to pH 7 thus suggesting that at low pH NPN is accommodated in a more hydrophobic environment. It has been previously demonstrated that AaegOBP1 undergoes a pH-dependent conformational change. Although AaegOBP1 does not bind MOP at low pH, it has higher affinity for the fluorescence reporter: Kd = 1.07 ± 0.15 μM, pH 5.5; Kd = 3.31 ± 0.48 μM, pH 7. Lack of binding to odorants at low pH has been observed with the Culex orthologous protein, CquiOBP124 and other OBPs, but insect fatty carriers bind ligands at low and high pH values38.\n\nEmission spectra at pH 5.5 (top traces) were considerably blue shifted relative to pH 7 (lower traces). Fluorescence intensity was also relatively higher at lower pH.\n\n\nConclusion\n\nA clear mechanistic explanation for the findings reported here must await further structural experimental data, particularly elucidation of crystal structures of AaegOBP1 bound to MOP and NPN separately as well as simultaneously. There are currently five structures of mosquito OBP1s deposited in Protein Data Bank (PDB), namely, AgamOBP1-PEG (PDB entry, 2ERB)21 (Figure 7A,B), AaegOBP1-PEG (3K1E)20, CquiOBP1-MOP (NMR, 2L2C; crystal, 3OGN)19 (Figure 7C,D), AgamOBP1-DEET (3N7H)18, AgamOBP1-sulcatone (4FQT)17. Unfortunately, the only OBP-NPN complex (3S0B)39 deposited in PDB is for an OBP from the European honey bee, AmelOBP14, which differs from classical OBPs for having two, instead of three, disulfide bridges. Here, NPN is bound in the central cavity of the protein. In CquiOBP1, MOP (Figure 1) has its long lipid tail bound to a hydrophobic tunnel formed between helices 4 and 5 (Figure 7D) and only its lactone/acetyl ester polar moiety is accommodated in part of the central cavity (Figure 7D, dashed circle). It is, therefore, feasible that MOP and NPN were bound simultaneously, and given the vicinity between the two ligands MOP could cause quenching of NPN fluorescence. It has been shown that in AgamOBP1 DEET is localized at the edge of the binding pocket in the equivalent hydrophobic tunnel that accommodates the lipid tail of MOP in CquiOBP1 (Figure 7D). Providing that NPN would bind in the central cavity, as in AmelOBP14, the distance between DEET and NPN would prevent quenching and, therefore, the “lack of binding” suggested by DEET titration (Figure 2) might be interpreted with caution. The unusual increase in fluorescence observed here might be explained at least in part by micelle formation. Unbound NPN, either displaced from AaegOBP1 or remaining in solution, could be housed in MOP-derived micelles and in this hydrophobic environment a blue shift and fluorescence increase are expected. It is also conceivable that at higher doses of MOP a second molecule of this ligand binds to AaegOBP1. There is another hydrophobic moiety bordered by helices α1 and α4 and occupied by PEG in the “apo-AgamOBP1”, which could possibly accommodate another ligand (Figure 7, highlighted with circles). If so, NPN could be accommodated in a more hydrophobic environment thus causing a blue shift and additional increase in fluorescence. This change in NPN environment could be triggered by a conformational change. Of notice, NPN fluorescence emission was blue shifted at acidic pH (5.5) compared to neutral pH (7) (Figure 6). Thus in the acidic conformation of AaegOBP1 NPN was more protected from the solvent, i.e., it is likely to be localized in a more hydrophobic environment. Previously, we have observed binding of two ligands to an insect OBP. The pheromone-binding protein from the silkworm moth, Bombyx mori, has been crystallized with two molecules of the bell pepper odorant, 2-isobutyl-3-methoxypyrazine40. Likewise, fatty acid binding proteins have been demonstrated to bind two molecules of the same ligand, oleic acid41. Recently, it has been suggested that DEET and NPN might bind simultaneously to AgamOBP117, but experimental evidence showing increase in NPN fluorescence and blue shift data was missing. The hypotheses put forward here on the basis of our findings must await experimental evidence, in particular X-ray crystallography studies. Studies to test these hypotheses may lead to more effective fluorescence reporters and a better understanding of OBP odorant binding.\n\n(A and C) Hydrophobicity surfaces of AaegOBP1 and CquiOBP1. (B and D) Ribbon displays of the same structures. A potential secondary binding site for MOP is highlighted with circles. It is occupied by PEG in AaegOBP1 but “empty” in CquiOBP1. The central cavity is highlighted in (D) with a dashed circle and shows that only the polar head (lactone moiety) of MOP is housed in the core of the protein. Figure prepared with UCSF Chimera software.\n\n\nData availability\n\nF1000Research: Dataset 1. Fluorescence reporter assay data with assessing binding of insect repellents to the yellow fever mosquito (Culex quinquefasciatus) odorant binding protein AaegOBP1, 10.5256/f1000research.5879.d3994842", "appendix": "Author contributions\n\n\n\nWSL designed the experiments. GML and WSL carried out the research. WSL analyzed the data and wrote the manuscript. All authors revised the manuscript and agreed to its final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported in part from unrestricted gifts from Fuji Flavor Co. and Bedoukian Research Inc.\n\n\nAcknowledgements\n\nWe thank Dr. Robert Bedoukian (Bedoukian Research Inc.) for MOP and PMD samples, and Dr. Kamal Chauhan (USDA, ARS) for picaridin and IR3535 samples. We are indebted to Mr. Garrison Buss (UC Davis), Drs. Yuko Ishida (Toyama University, Japan), Julien Pelletier (Keele University, UK), Pingxi Xu (UC Davis) and David Wilson (UC Davis) for comments and suggestion to improve an earlier version of the manuscript.\n\n\nReferences\n\nClyne PJ, Warr CG, Freeman MR, et al.: A novel family of divergent seven-transmembrane proteins: candidate odorant receptors in Drosophila. Neuron. 1999; 22(2): 327–38. PubMed Abstract \| Publisher Full Text\n\nGao Q, Chess A: Identification of candidate Drosophila olfactory receptors from genomic DNA sequence. Genomics. 1999; 60(1): 31–9. PubMed Abstract \| Publisher Full Text\n\nVosshall LB, Amrein H, Morozov PS, et al.: A spatial map of olfactory receptor expression in the Drosophila antenna. Cell. 1999; 96(5): 725–36. PubMed Abstract \| Publisher Full Text\n\nLeal WS: Odorant reception in insects: roles of receptors, binding proteins, and degrading enzymes. Annu Rev Entomol. 2013; 58: 373–91. PubMed Abstract \| Publisher Full Text\n\nBenton R, Sachse S, Michnick SW, et al.: Atypical membrane topology and heteromeric function of Drosophila odorant receptors in vivo. PLoS Biol. 2006; 4(2): e20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVogt RG, Riddiford LM: Pheromone binding and inactivation by moth antennae. Nature. 1981; 293(5828): 161–3. PubMed Abstract \| Publisher Full Text\n\nIshida Y, Cornel AJ, Leal WS: Identification and cloning of a female antenna-specific odorant-binding protein in the mosquito Culex quinquefasciatus. J Chem Ecol. 2002; 28(4): 867–71. PubMed Abstract \| Publisher Full Text\n\nBohbot J, Pitts RJ, Kwon HW, et al.: Molecular characterization of the Aedes aegypti odorant receptor gene family. Insect Mol Biol. 2007; 16(5): 525–37. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarey AF, Wang G, Su CY, et al.: Odorant reception in the malaria mosquito Anopheles gambiae. Nature. 2010; 464(7285): 66–71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeal WS, Choo YM, Xu P, et al.: Differential expression of olfactory genes in the southern house mosquito and insights into unique odorant receptor gene isoforms. Proc Natl Acad Sci U S A. 2013; 110(46): 18704–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang G, Carey AF, Carlson JR, et al.: Molecular basis of odor coding in the malaria vector mosquito Anopheles gambiae. Proc Natl Acad Sci U S A. 2010; 107(9): 4418–23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHughes DT, Pelletier J, Luetje CW, et al.: Odorant receptor from the southern house mosquito narrowly tuned to the oviposition attractant skatole. J Chem Ecol. 2010; 36(8): 797–800. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPelletier J, Hughes DT, Luetje CW, et al.: An odorant receptor from the southern house mosquito Culex pipiens quinquefasciatus sensitive to oviposition attractants. PLoS One. 2010; 5(4): e10090. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhu F, Xu P, Barbosa RM, et al.: RNAi-based demonstration of direct link between specific odorant receptors and mosquito oviposition behavior. Insect Biochem Mol Biol. 2013; 43(10): 916–23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXu P, Choo YM, De La Rosa A, et al.: Mosquito odorant receptor for DEET and methyl jasmonate. Proc Natl Acad Sci U S A. 2014; 111(46): 16592–16597. PubMed Abstract \| Publisher Full Text\n\nMcBride CS, Baier F, Omondi AB, et al.: Evolution of mosquito preference for humans linked to an odorant receptor. Nature. 2014; 515(7526): 222–7. PubMed Abstract \| Publisher Full Text\n\nMurphy EJ, Booth JC, Davrazou F, et al.: Interactions of Anopheles gambiae odorant-binding proteins with a human-derived repellent: implications for the mode of action of n,n-diethyl-3–methylbenzamide (DEET). J Biol Chem. 2013; 288(6): 4475–85. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTsitsanou KE, Thireou T, Drakou CE, et al.: Anopheles gambiae odorant binding protein crystal complex with the synthetic repellent DEET: implications for structure-based design of novel mosquito repellents. Cell Mol Life Sci. 2012; 69(2): 283–97. PubMed Abstract \| Publisher Full Text\n\nMao Y, Xu X, Xu W, et al.: Crystal and solution structures of an odorant-binding protein from the southern house mosquito complexed with an oviposition pheromone. Proc Natl Acad Sci U S A. 2010; 107(44): 19102–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeite NR, Krogh R, Xu W, et al.: Structure of an odorant-binding protein from the mosquito Aedes aegypti suggests a binding pocket covered by a pH-sensitive “Lid”. PLoS One. 2009; 4(11): e8006. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWogulis M, Morgan T, Ishida Y, et al.: The crystal structure of an odorant binding protein from Anopheles gambiae: evidence for a common ligand release mechanism. Biochem Biophys Res Commun. 2006; 339(1): 157–64. PubMed Abstract \| Publisher Full Text\n\nBiessmann H, Andronopoulou E, Biessmann MR, et al.: The Anopheles gambiae odorant binding protein 1 (AgamOBP1) mediates indole recognition in the antennae of female mosquitoes. PLoS One. 2010; 5(3): e9471. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPelletier J, Guidolin A, Syed Z, et al.: Knockdown of a mosquito odorant-binding protein involved in the sensitive detection of oviposition attractants. J Chem Ecol. 2010; 36(3): 245–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeal WS, Barbosa RM, Xu W, et al.: Reverse and conventional chemical ecology approaches for the development of oviposition attractants for Culex mosquitoes. PLoS One. 2008; 3(8): e3045. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLagarde A, Spinelli S, Qiao H, et al.: Crystal structure of a novel type of odorant-binding protein from Anopheles gambiae, belonging to the C-plus class. Biochem J. 2011; 437(3): 423–30. PubMed Abstract \| Publisher Full Text\n\nQiao H, He X, Schymura D, et al.: Cooperative interactions between odorant-binding proteins of Anopheles gambiae. Cell Mol Life Sci. 2011; 68(10): 1799–813. PubMed Abstract \| Publisher Full Text\n\nIovinella I, Bozza F, Caputo B, et al.: Ligand-binding study of Anopheles gambiae chemosensory proteins. Chem Senses. 2013; 38(5): 409–19. PubMed Abstract \| Publisher Full Text\n\nLeal WS, Chen AM, Ishida Y, et al.: Kinetics and molecular properties of pheromone binding and release. Proc Natl Acad Sci U S A. 2005; 102(15): 5386–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBan L, Zhang L, Yan Y, et al.: Binding properties of a locust’s chemosensory protein. Biochem Biophys Res Commun. 2002; 293(1): 50–4. PubMed Abstract \| Publisher Full Text\n\nBan L, Scaloni A, Brandazza A, et al.: Chemosensory proteins of Locusta migratoria. Insect Mol Biol. 2003; 12(2): 125–34. PubMed Abstract \| Publisher Full Text\n\nIshida Y, Chen AM, Tsuruda JM, et al.: Intriguing olfactory proteins from the yellow fever mosquito, Aedes aegypti. Naturwissenschaften. 2004; 91(9): 426–31. PubMed Abstract \| Publisher Full Text\n\nZhou JJ, He XL, Pickett JA, et al.: Identification of odorant-binding proteins of the yellow fever mosquito Aedes aegypti: genome annotation and comparative analyses. Insect Mol Biol. 2008; 17(2): 147–63. PubMed Abstract \| Publisher Full Text\n\nZhou JJ, He XL, Pickett JA, et al.: Corrigendum. Insect Mol Biol. 2008; 17(4): 445. Publisher Full Text\n\nWojtasek H, Leal WS: Conformational change in the pheromone-binding protein form Bombyx mori induced by pH and by interaction with membrane. J Biol Chem. 1999; 274(43): 30950–6. PubMed Abstract \| Publisher Full Text\n\nLeal WS: Duality monomer-dimer of the pheromone-binding protein from Bombyx mori. Biochem Biophys Res Commun. 2000; 268(2): 521–9. PubMed Abstract \| Publisher Full Text\n\nLautenschlager C, Leal WS, Clardy J: Coil-to-helix transition and ligand release of Bombyx mori pheromone-binding protein. Biochem Biophys Res Commun. 2005; 335(4): 1044–50. PubMed Abstract \| Publisher Full Text\n\nLaurence BB, Pickett JA: erythro-6-Acetoxy-5-hexadecanolide, the major component of a mosquito oviposition attractant pheromone. J Chem Soc Chem Commun. 1982; 1982(1): 59–60. Publisher Full Text\n\nIshida Y, Ishibashi J, Leal WS: Fatty acid solubilizer from the oral disk of the blowfly. PLoS One. 2013; 8(1): e51779. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSpinelli S, Lagarde A, Iovinella I, et al.: Crystal structure of Apis mellifera OBP14, a C-minus odorant-binding protein, and its complexes with odorant molecules. Insect Biochem Mol Biol. 2012; 42(1): 41–50. PubMed Abstract \| Publisher Full Text\n\nLautenschlager C, Leal WS, Clardy J: Bombyx mori pheromone-binding protein binding nonpheromone ligands: implications for pheromone recognition. Structure. 2007; 15(9): 1148–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCai J, Lucke C, Chen Z, et al.: Solution structure and backbone dynamics of human liver fatty acid binding protein: fatty acid binding revisited. Biophys J. 2012; 102(11): 2585–94. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeal GM, Leal SW: Dataset 1 in ‘Binding of a fluorescence reporter and a ligand to an odorant-binding protein of the yellow fever mosquito, Aedes aegypti’. F1000Research. 2014. Data Source" }	[ { "id": "7040", "date": "29 Dec 2014", "name": "Kostas Iatrou", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript describes interesting results obtained in the course of what of an investigation initially designed as a high school project undertaken in order to deduce whether AaegOBP1, an odorant binding protein of the yellow fever mosquito Aedes aegypti, binds DEET and/or other known mosquito repellents such as icaridin, PMD and IR3535. The relevant experiments consisted of classical binding competition assays by the tested repellents against an AaegOBP1 pre-bound fluorescent reporter molecule, NPN, causing reductions in NPN-emitted fluorescent quenching with the latter serving as measure of mosquito repellent binding to AaegOBP1 resulting in displacement of the pre-bound NPN. While the experiments suggested that the specific OBP may only bind the tested repellents with limited affinity relative to NPN, they also produced results that could not have been predicted a priori. The first concerned an unexpected property of a mosquito (Culex quiquefasciatus) oviposition pheromone (MOP) that was used as positive control for binding to AaegOBP1. Thus, while titration AaegOBP1/NPN complexes by increasing quantities of MOP produced the anticipated reduction in NPN fluorescence, titrations with higher MOP doses led to gradual increases of fluorescence emitted by NPN accompanied by a wavelength shift toward the blue region of the spectrum. To explain this finding as well as the parallel observation that the same phenomenon also occurs at the same MOP concentrations in the absence of AaegOBP1, the authors have postulated the formation of MOP micelles forming a highly hydrophobic environment to which displaced and free NPN may bind. The second intriguing finding has been that at a low pH of 5.5 at which AaegOBP1 is unable to bind MOP, this protein binds NPN with higher affinity relative to a neutral pH, causing higher emitted fluorescence with a concomitant blue-shift in the emission wavelength suggestive of the formation of a higher hydrophobicity environment to which NPN is bound. Based on these findings as well as the crystal structures of CquiOBP1 and Anopheles gambiae AgamOBP1, both AaegOBP1 orthologs, in complex with MOP and DEET, respectively, as well as the complex of the honey bee AmelOBP14 with NPN, the authors postulate the possibility that NPN and MOP could bind simultaneously to AaegOBP1 at a neutral pH. In turn, this possibility suggests that caution should also be exercised for the postulated conclusion regarding the low affinity binding of DEET to AaegOBP1, because DEET binding to a separate pocket might not necessarily result in displacement of NPN. Suggestions:For the first set of observations related to the postulated micelle formation by MOP at concentrations of 12.5 μM or higher, the hypothesized explanation is quite reasonable. A dynamic light scattering experiment using MOP in buffer alone could further strengthen the postulated hypothesis. Moreover, a NPN titration experiment similar to that shown in Fig. 5B but at a pH 5.5, which should result in protonation e.g. of the acetoxy-group of MOP, could reveal whether an increase in micelle size occurs or not. This latter experiment could also provide additional suggestive evidence for the postulated creation of a more hydrophobic environment for NPN binding in AaegOBP1 at the acidic pH.For the structural considerations presented in the conclusions, as the authors indicate, co-crystalization of AaegOBP1 with NPN, MOP or both, will be required in order for conclusive interpretations to be drawn. Nevertheless, it is not clear to us why in a case of simultaneous binding of NPN and MOP (AaegOBP1-MOP-NPN complex), NPN should move to a different binding pocket of higher hydrophobicity producing higher fluorescence emission and a blue shift, only at higher MOP concentrations and not at lower ones. If, on the other hand, the requirement for higher MOP concentrations is interpreted as indicative of the formation of AaegOBP1-MOP-NPN-MOP complexes, a docking model should indicate whether enough space exists in the L-shaped tunnel of the AaegOBP1 monomer for simultaneous binding of 3 molecules. For the low apparent affinity of AaegOBP1 for DEET, it is indeed possible that the binding of DEET and NPN to AaegOBP1 are not mutually exclusive, hence the low reduction in emitted NPN fluorescence in the presence of increasing concentrations of DEET. A docking model should indicate whether the possibility of nearby binding sites or even overlapping ones for NPN and DEET is predicted, which would lead to fluorescence quenching rather than reduction due to NPN displacement.Finally, the authors should provide a concluding statement as to whether and how these interesting findings relate to the contributions of OBPs in the mosquito's olfactory function under normal conditions.", "responses": [ { "c_id": "1155", "date": "02 Jan 2015", "name": "Walter Leal", "role": "Author Response", "response": "First of all, we would like to thank you for the time and effort to process and evaluate our article. We were delighted to read the laudatory comments in your report. We have considered carefully your suggestions (thank you very much), and performed an additional experiment, which will be incorporated in Figure 5C. Specifically, we performed NPN titration with MOP in ammonium acetate buffer, pH 5.5. Although we agree that the suggested docking experiments (bullets 2 and 3) might add to the discussion, ultimately the structural hypotheses raised in the article shall be supported or refuted by X-ray crystallography-based structural evidence. While none of the authors is well versed in molecular modeling, we have the expertise and collaboration in place to rigorously test the structural hypotheses. In collaboration with our UCD colleague, Dr. David Wilson, Gabriel has been able to crystallize other OBPs, and we are confident that he/we will succeed in crystalizing AaegOBP1 complexes and address these questions. Obviously, time is uncertain in crystallography and the scope of the new work seems to belong to future publication(s). In the revised version of our F1000Research article, we will add comments regarding contributions of OBPs to mosquito olfaction, as suggested. Additionally, we will add results shown further evidence of micelle formation even at low pH, except that the effect was manifested at slightly higher concentrations of MOP. In sum, figures B and C are almost identical, but the blue shift and fluorescence increase were clearly observed starting at 15 and 17.5 uM at pH 5.5 (revised Fig. 5C) as compared to 12.5 and 15 uM (Fig. 5B). We do hope that now the article meets your approval. Sincerely, WSL & GML" } ] }, { "id": "7176", "date": "02 Jan 2015", "name": "Paolo Pelosi", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe phenomenon described in this paper is well known and documented in many papers. However, it has never been directly examined and explained in detailed. Therefore, it is nice and useful to have a focused study to describe and dissect such apparently anomalous behaviour once and for all. I fully agree with the Authors that the formation of micelles is the most likely explanation. We have come across this same phenomenon several times and I have always discussed this fact with my students hypothesising the formation of micelles as the most likely reason behind this. A brief explanation of some anomalous binding curves can also be found in some of our published papers, most recently in Sun et al. (2012). When a ligand capable of forming micelles also has affinity for the protein, we observe a decrease of fluorescence, followed by an increase when titrating the protein (the U curve observed in this paper). When the ligand has poor affinity for the protein, we only observe a constant increase in fluorescence. Sometimes we have also recorded a complex behaviour: the intensity of fluorescence experiences an increase at low concentration values of the ligand, then drops when more ligand is added. In this case, the phenomenon could be explained by assuming that the ligand enters the binding pocket without displacing the fluorescent probe. As the Authors point out such facts can occur and have been documented with OBPs and CSPs. The increase of fluorescence in such case would be the result of the increased hydrophobicity of the binding pocket due to the presence of a ligand, usually a highly hydrophobic molecule, as in the case of many pheromones of Lepidoptera and Diptera. As the concentration of the ligand increases, competition with bound 1-NPN can take over producing a decrease in fluorescence.This study could be complemented (but not necessarily) by monitoring the intrinsic fluorescence of the tryptophan, which appropriately is located inside the binding pocket of the protein. Particularly in the case of DEET, which is an aromatic compound, if the molecule binds to OBP without being able to displace 1-NPN, we should observe a strong quenching of the tryptophan fluorescence. Overall the paper is well written and the observed phenomenon clearly described and explained. I have only one minor concern:The Authors report the emission spectrum of 1-NPN alone (in Tris buffer at pH 7.4) with a maximum around 470 nm and in the presence of protein at about 440. In my experience, I found that 1-NPN in buffer produces a peak with a maximum at 480, which is shifted in the presence of a binding protein to values generally between 400 and 410 nm, in some cases even below 400. This has been observed with a large number of OBPs, including some of mosquitoes, although not with the specific OBP used in this study. I suggest that the Authors double check these data, also in relationship to the instrument calibration.", "responses": [ { "c_id": "1156", "date": "05 Jan 2015", "name": "Walter Leal", "role": "Author Response", "response": "We appreciate your time and effort to evaluate our article, and are delighted to hear that it meets your approval. We will certainly cite Sun et al. (2012) in the revised version of the article. The apparent formation of (E)-ß-farnesene micelle, as shown in your Figure 5, skipped our attention, perhaps because this phenomenon has never been examined and explained in detail. We will certainly give the appropriate credit in the revised version. Regarding your minor concern, we have verified wavelength accuracy per vendor’s instruction manual. We are, therefore, confident that the data set reported is accurate. It is worth pointing out, however, that apparent discrepancies may be explained at least in part by the proteins studied. For AaegOBP1, the NPN peaks at pH values 7 and 5 are quite different (our Figure 6), but in the presence of CquiOBP1 (Figure 5 in Leal et al, 2008) or AfunOBP1 (Figure 9, Xu et al., 2010) the wavelength for the peaks at high and low pH are nearly the same, i.e., ca. 400 nm. All these studies were performed in our laboratory with the same instrument. Again, thank you very much for your suggestions. Sincerely, WSL & GML" } ] } ]	1	https://f1000research.com/articles/3-305
https://f1000research.com/articles/3-271/v1	12 Nov 14	{ "type": "Research Note", "title": "The Open Science Peer Review Oath", "authors": [ "Jelena Aleksic", "Adrian Alexa", "Teresa K Attwood", "Neil Chue Hong", "Martin Dahlö", "Robert Davey", "Holger Dinkel", "Konrad U Förstner", "Ivo Grigorov", "Jean-Karim Hériché", "Leo Lahti", "Dan MacLean", "Michael L Markie", "Jenny Molloy", "Maria Victoria Schneider", "Camille Scott", "Richard Smith-Unna", "Bruno Miguel Vieira", "as part of the AllBio: Open Science & Reproducibility Best Practice Workshop", "Jelena Aleksic", "Adrian Alexa", "Teresa K Attwood", "Neil Chue Hong", "Martin Dahlö", "Robert Davey", "Holger Dinkel", "Konrad U Förstner", "Ivo Grigorov", "Jean-Karim Hériché", "Leo Lahti", "Michael L Markie", "Jenny Molloy", "Maria Victoria Schneider", "Camille Scott", "Richard Smith-Unna", "Bruno Miguel Vieira" ], "abstract": "One of the foundations of the scientific method is to be able to reproduce experiments and corroborate the results of research that has been done before. However, with the increasing complexities of new technologies and techniques, coupled with the specialisation of experiments, reproducing research findings has become a growing challenge. Clearly, scientific methods must be conveyed succinctly, and with clarity and rigour, in order for research to be reproducible. Here, we propose steps to help increase the transparency of the scientific method and the reproducibility of research results: specifically, we introduce a peer-review oath and accompanying manifesto. These have been designed to offer guidelines to enable reviewers (with the minimum friction or bias) to follow and apply open science principles, and support the ideas of transparency, reproducibility and ultimately greater societal impact. Introducing the oath and manifesto at the stage of peer review will help to check that the research being published includes everything that other researchers would need to successfully repeat the work. Peer review is the lynchpin of the publishing system: encouraging the community to consciously (and conscientiously) uphold these principles should help to improve published papers, increase confidence in the reproducibility of the work and, ultimately, provide strategic benefits to authors and their institutions. Future incarnations of the various national Research Excellence Frameworks (REFs) will evolve away from simple citations towards measurable societal value and impact. The proposed manifesto aspires to facilitate this goal by making transparency, reproducibility and citizen-scientist engagement (with the knowledge-creation and dissemination processes) the default parameters for performing sound research.", "keywords": [ "An essential part of the scientific method is that researchers can repeat the experiments of others and test the outcomes themselves. To achieve this requires accurate reporting not just of the results of those experiments but also of the methods that underpin them. However", "as science becomes more technology-driven", "the equipment used is more specialised", "the data generated is harder to represent in traditional media", "and reporting how experiments were performed so that independent researchers can repeat them gets progressively harder. Reproducibility in science is a hot topic and a concerning one", "indeed", "several commentators have concluded that fallibilities in the way that research investigations are currently conducted", "and how their results are disseminated via article publication have become detrimental to the scientific process1–4. The difficulties in ensuring reproducibility are multi-faceted: the problems are systemic. Policy makers", "funding agencies", "academic institutions", "scientific publishers", "scientists themselves and the vehicles through which they publish each contribute to a complicated web of issues that conspire against the publication of reproducible results5. Various measures have been proposed to try to combat these problems", "ranging from top-down strategies through government initiatives6", "to bottom-up strategies such as providing checks and balances for research integrity during the publishing process7. Measures like this tend to come with their own problems and", "in some cases", "can provide further barriers to reproducibility8." ], "content": "Introduction\n\nAn essential part of the scientific method is that researchers can repeat the experiments of others and test the outcomes themselves. To achieve this requires accurate reporting not just of the results of those experiments but also of the methods that underpin them. However, as science becomes more technology-driven, the equipment used is more specialised, the data generated is harder to represent in traditional media, and reporting how experiments were performed so that independent researchers can repeat them gets progressively harder. Reproducibility in science is a hot topic and a concerning one; indeed, several commentators have concluded that fallibilities in the way that research investigations are currently conducted, and how their results are disseminated via article publication have become detrimental to the scientific process1–4. The difficulties in ensuring reproducibility are multi-faceted: the problems are systemic. Policy makers, funding agencies, academic institutions, scientific publishers, scientists themselves and the vehicles through which they publish each contribute to a complicated web of issues that conspire against the publication of reproducible results5. Various measures have been proposed to try to combat these problems, ranging from top-down strategies through government initiatives6, to bottom-up strategies such as providing checks and balances for research integrity during the publishing process7. Measures like this tend to come with their own problems and, in some cases, can provide further barriers to reproducibility8.\n\nOne way in which reproducibility issues can be tackled is through the implementation of open science and open data practices9,10. As attendees of the AllBio: Open Science & Reproducibility Best Practice Workshop, we discussed how principles of open science could be instilled into the current research workflow; as part of this debate, we tried to identify ways in which reproducibility might be improved.\n\nOne route into this workflow is through the peer review process. Peer review is an important gatekeeper and a key part of scientific discourse. Before any research findings can be formally accepted, they must be evaluated and commented upon by peers (experts in their fields), who then provide advice about the quality or validity of the work to Editors, or in the case of open peer review and post-publication invited peer review systems, to the readers themselves. Importantly, peer review happens at a personal rather than institutional level and is carried out by individuals; it is therefore an ideal mechanism for getting a message across to the majority of researchers given everyone peer reviews or is peer reviewed. Of course, the peer-review process is not infallible11,12. The issues are many and varied, including the time available to perform thorough reviews, reviewers’ expertise, journals’ perception of relevance/interest/impact, and so on. Arguably, one of the most significant problems – certainly the one that generates most friction – is that reviewers can safely dispense self-serving and biased critiques, fully protected by the mask of anonymity.\n\nScientists have become sufficiently frustrated by these issues to devise ad hoc solutions to help safeguard the quality of reviews and allow reviewers to affirm that they will review in an ethical and professional way, and encourage clearer review processes. This has led to the articulation of various forms of reviewer’s oath (e.g. 13–15). It is these that inspired us. Building on this work, we have formulated an oath that codifies the role of reviewers in helping to ensure that the science they review is sufficiently open and reproducible; it includes guidelines not just on how to review professionally, but also on how to support transparent, reproducible and responsible research, while optimising its societal impact and maximising its visibility. We suggest a mode of constructive dialogue between respectful individuals.\n\nThe new oath is accompanied by a manifesto that develops the principles set out in the guidelines, and provides further direction for upholding responsible and interactive reviews, as well as the necessary information for other researchers to reproduce the results. A key tenet is that the oath is not meant to be burdensome or to cause friction between reviewers and authors; in fact, their cooperation could improve the accuracy of reviews16. The goal is to provide a supportive framework for guiding reviewers toward professional and ethical behaviours, and to provide the necessary checks on whether they would be able to reproduce the work. If the issue of reproducibility can be satisfied at the point of peer review, then published results should be more reliable, and the scientific community can have greater faith that what they read is solid enough to build on.\n\n\nThe Open Science Reviewer’s Oath\n\nThe oath is a simple checklist to use when reviewing or considering a review request. We recommend that reviewers add a link to this oath (Box 1) at the top of each review as they begin, in order to provide an aide memoire to open review practice, and to inform the authors and potential publishers of the work of their intentions. We hope that by being explicit about the intent, the review will seem less like a cloak-and-dagger process, it will make constructive criticism easier for the author to receive and for the reviewer to provide, and it will also help to spread the practice of open reviewing.\n\n\n\ni) I will sign my review in order to be able to have an open dialogue with you\n\nii) I will be honest at all times\n\niii) I will state my limits\n\niv) I will turn down reviews I am not qualified to provide\n\nv) I will not unduly delay the review process\n\nvi) I will not scoop research that I had not planned to do before reading the manuscript\n\nvii) I will be constructive in my criticism\n\nviii) I will treat reviews as scientific discourses\n\nix) I will encourage discussion, and respond to your and/or editors’ questions\n\nx) I will try to assist in every way I ethically can to provide criticism and praise that is valid, relevant and cognisant of community norms\n\nxi) I will encourage the application of any other open science best practices relevant to my field that would support transparency, reproducibility, re-use and integrity of your research\n\nxii) If your results contradict earlier findings, I will allow them to stand, provided the methodology is sound and you have discussed them in context\n\nxiii) I will check that the data, software code and digital object identifiers are correct, and the models presented are archived, referenced, and accessible\n\nxiv) I will comment on how well you have achieved transparency, in terms of materials and methodology, data and code access, versioning, algorithms, software parameters and standards, such that your experiments can be repeated independently\n\nxv) I will encourage deposition with long-term unrestricted access to the data that underpin the published concept, towards transparency and re-use\n\nxvi) I will encourage central long-term unrestricted access to any software code and support documentation that underpin the published concept, both for reproducibility of results and software availability\n\nxvii) I will remind myself to adhere to this oath by providing a clear statement and link to it in each review I write, hence helping to perpetuate good practice to the authors whose work I review.\n\n\nThe manifesto\n\nEach point of the reviewer’s oath relates to open principles that we consider important; the collection of these principles is the manifesto. The manifesto relates to the oath as follows:\n\ni) I will sign my review in order to be able to have an open dialogue with you\n\nI recognise that reviewing is a role that gives me advantage over you and that anonymity allows abuse of your trust. I will not do this.\n\nii) I will be open and honest at all times\n\niii) I will state my limits\n\niv) I will turn down reviews I am not qualified to provide\n\nv) I will not unduly delay the review process\n\nvi) I will not scoop research that I had not planned to do before reading the manuscript\n\nI recognise that integrity is a social act that requires the majority to hold shared convictions; I will use the majority of ‘doves’ to balance the ‘hawks’ in my review by sharing the content.\n\nI will always state the boundaries of my scientific knowledge and practice; I openly acknowledge that I am not an expert in, and cannot satisfactorily assess every aspect of, my field. I will inform you and the journal when this situation arises.\n\nI will not always be an appropriate reviewer. I will provide journal editors with a fair assessment of my ability and, when necessary, decline to review, and will always expand on the reasons.\n\nI will not write a negative review with the intention of blocking publication or delaying publication. In the case where I have already come to the same (or different) conclusions from the author I will state this fact and suggest the possibility of cooperative publication (either back-to-back) or merge a paper.\n\nI understand that there are conflicts in my field. Sometimes, there may be good reasons for remaining anonymous, which may relate to the integrity of others. Wherever possible, I will highlight abuses of integrity and turn down invitations if I feel I have such a direct conflict that would inappropriately affect my review.\n\nvii) I will be constructive in my criticism\n\nviii) I will treat reviews as scientific discourses\n\nix) I will encourage discussion, and respond to your and/or editors’ questions\n\nI will happily engage in conversation with you about your work, providing constructive criticism where appropriate.\n\nx) I will try to assist in every way I ethically can to get your manuscript published, by providing criticism and praise that is valid, relevant and cognisant of community norms\n\nxi) I will encourage the application of any other open science best practices relevant to my field that would support transparency, reproducibility, re-use and integrity of your research\n\nxii) If your results contradict earlier findings, I will allow them to stand, provided the methodology is sound and that you have discussed them in context\n\nxiii) I will check that the data, software code and digital object identifiers are correct, and the models presented are archived, referenced, and accessible\n\nxiv) I will comment on how well you have achieved transparency, in terms of materials and methodology, data and code access, versioning, algorithms, software parameters and standards, so that your experiments can be repeated independently\n\nxv) I will encourage deposition with long-term unrestricted access to the data that underpin the published concept, towards transparency and re-use;\n\nxvi) I will encourage central long-term unrestricted access to any software code and support documentation that underpin the published concept, both for reproducibility of results and software availability\n\nI will uphold and advocate open science practice by pointing out where I believe that the authors can do better with respect to deposition of data, citation of accessions and code etc. Often this will mean circumventing current norms.\n\nxvii) I will remind myself to adhere to this oath by providing a clear statement and link to it in each review I write, helping to perpetuate good practice to the authors whose work I review.\n\nAs part of my role as a scientist and an open reviewer, I will help other reviewers when they need guidance or support. I understand that new reviewers may not feel entirely secure in managing the conflicts that often arise from the normal academic process. In these cases I will judge a review on its merit and not the individual who has written it.", "appendix": "Author contributions\n\n\n\nDan Maclean, Ivo Grigorov, Michael Markie, Teresa Attwood, Konrad Förstner, Jean-Karim Heriche and Neil Chue Hong conceived and designed the oath and prepared the first draft of the manuscript. All the other authors in the working group were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nMM is currently employed by F1000Research. His role at the journal does not include any involvement in the pre-publication editorial checks, or with the refereeing process.\n\n\nGrant information\n\nALLBIO - Broadening the Bioinformatics Infrastructure to unicellular, animal, and plant science, Project reference: 289452, Funded under: FP7-KBBE. We would also like to thank The Genome Analysis Centre (TGAC, Norwich, UK) and the Biotechnology and Biological Sciences Research Council (BBSRC, UK). IG was funded by FP7 FOSTER (Grant 612 425).\n\n\nAcknowledgements\n\nWe would like to thank The Genome Analysis Centre (TGAC, Norwich, UK) for organising and hosting the workshop.\n\nWe would also like to thank Peter Murray Rust for comments on the preprint (https://zenodo.org/record/12273) and contributing an additional principle to the oath.\n\n\nReferences\n\nIoannidis JP: Why most published research findings are false. PLoS Med. 2005; 2(8): e124. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIoannidis JP, Allison DB, Ball CA, et al.: Repeatability of published microarray gene expression analyses. Nat Genet. 2009; 41(2): 149–55. PubMed Abstract \| Publisher Full Text\n\nPrinz F, Schlange T, Asadullah K: Believe it or not: how much can we rely on published data on potential drug targets? Nat Rev Drug Discov. 2011; 10(9): 712. PubMed Abstract \| Publisher Full Text\n\nHines WC, Su Y, Kuhn I, et al.: Sorting out the FACS: a devil in the details. Cell Rep. 2014; 6(5): 779–81. PubMed Abstract \| Publisher Full Text\n\nCollins FS, Tabak LA: Policy: NIH plans to enhance reproducibility. Nature. 2014; 505(7485): 612–3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEuropean Commission Responsible Research & Innovation Policy. 2012. Reference Source\n\nIorns E, Chong C: New forms of checks and balances are needed to improve research integrity [v1; ref status: indexed, http://f1000r.es/32k]. F1000Res. 2014; 3: 119. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStodden V: Changes in the Research Process Must Come From the Scientific Community, not Federal Regulation. 2013. Reference Source\n\nMolloy JC: The Open Knowledge Foundation: open data means better science. PLoS Biol. 2011; 9(12): e1001195. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPereira S, Gibbs RA, McGuire AL: Open access data sharing in genomic research. Genes (Basel). 2014; 5(3): 739–747. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPatel J: Why training and specialization is needed for peer review: a case study of peer review for randomized controlled trials. BMC Med. 2014; 12(1): 128. PubMed Abstract \| Publisher Full Text\n\nGlen AS: A New “Golden Rule” for Peer Review? Bull Ecol Soc Am. 2014; 95(4): 431–434. Publisher Full Text\n\nWatson M: The reviewers oath. 2013. Reference Source\n\nAlexander S: The Peer Reviewer’s Oath. 2014. Reference Source\n\nVerger A: My Reviewer Oath. 2014. Reference Source\n\nLeek JT, Taub MA, Pineda FJ: Cooperation between referees and authors increases peer review accuracy. PLoS One. 2011; 6(11): e26895. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "6804", "date": "25 Nov 2014", "name": "Vitaly Citovsky", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article addresses a very important issue of peer review. Although, many of us tend to regard it in a way of \"if it ain't broke, don't fix it\", we also are often frustrated with the process and wish it were more fair, transparent and to the point (rather than requesting numerous experiments that are only tangential to the paper's main message). Thus, constructive approach to potential improvement of the peer review system is always welcome and should be encouraged. With this in mind, I support, in principle, publication of this manuscript. On the other hand, the way that the authors present their ideas is not perfect. Here is why:First they make a huge emphasis in their introductory remarks on experimental reproducibility and societal impact. However, their \"oath\" does not address these issues specifically and constructively; instead, it offers declarative statements which are mostly trivial and already represent part and parcel of today's peer review process (e.g., \"I will comment on how well you have achieved transparency, in terms of materials and methodology, data and code access, versioning, algorithms, software parameters and standards, such that your experiments can be repeated independently\"). Second, the authors do not address clearly one very important aspect of potential improvement of the review process which is \"To request further experiments only as a last resort, and only if they are essential to validate the conclusions of the paper. No experiments extending the study beyond its conclusions, or with unreasonable cost or time implications, should be proposed. An estimate of time required for the additional work should be provided.\" This is a quote from Mariann Bienz and Kathy Weston who also addressed the issue of peer reviewing (http://elifesciences.org/elife-news/a-reviewers-charter).Third, the \"oath\" is unreasonably long. Again, I suggest to look at the post by Bienz and Weston, which unlike the present paper, is much clearer, laconic and to the point.Fourth, the authors quite nonchalantly suggest to overturn the cornerstone of the present review system - its anonymity. What is the chance for that? The reason for such a step should be well reasoned and substantiated.", "responses": [ { "c_id": "1171", "date": "12 Jan 2015", "name": "Dan MacLean", "role": "Author Response", "response": "Thanks Vitaly for your very helpful and candid report. We have now explained what we hope to achieve from the oath, how we think it could address the issue of reproducibility and how the oath could have a positive societal impact in the scientific community. We believe at the researcher level there is a unique opportunity to help spread open science practices when reviewing articles and help improve what the community as a whole believes should be available in a paper in order for it to be reproduced. We agree with your point about not asking for additional experiments, however we have decided to concentrate fully on elucidating the requirements of the open science principle specifically and not the other principles. As with the other reviewers and comments we have taken all of your advice and shortened the oath to make it simpler for reuse. We also agree with your point about anonymity and we have discussed this in the manuscript on why we should move towards open peer review." } ] }, { "id": "6797", "date": "26 Nov 2014", "name": "Christopher Chambers", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall, I believe this is a laudable proposal for a code of practice in academic peer review. In one sense it would be nice if such a code was unnecessary; after all, most of the practices outlined here should form part of any graduate training in science. In practice, of course, we know that the reality of peer review often falls short of achieving its aims. Here the authors outline 5 key principles and 17 key practices, which - if adhered to - would likely result in a more transparent and effective peer review mechanism.While I am supportive of this initiative, I do have some concerns about the way it is presented and I also wonder how adhering to it could be incentivised. I will outline below some suggestions for possible improvement:The authors begin by outlining the 17 key practices and only at the end do they group them together within governing principles. I think the overall structure of the paper would be clearer if this order were reversed - beginning with the five principles, explaining the key practices in each case that serve them, before returning to the code of practice or \"oath\" as the authors call it. Some of the individual practices in the oath are somewhat ambiguous to me, and others seem to overlap. For instance, what exactly does it mean for a reviewer to \"state their limits\"? This is elaborated later under principle 2 but I would recommend clearer descriptions at all times. Other practices seem very similar, or least belong to common subsets of behaviour; e.g. practice 7 and practice 10; practice 2 and 6; practices 13, 14, 15 and 16 are all very similar and not clearly distinguished. With some careful attention I suspect the number of practices here could be halved, which would improve readability and likely uptake. The language expanding on some of the core principles is perhaps little purple in places, e.g. \"I will use the majority of ‘doves’ to balance the ‘hawks’ in my review by sharing the content.\" I'm not entirely sure what this means, but if it refers to the intention to balance sharp critical feedback with constructive suggestions or positive feedback, this could perhaps we said in a more straightforward way. The authors should bear in mind that not all readers will have English as a first language, and so more direct and less metaphorical phrasing could widen the appeal. Two concerns with signed reviews need to be addressed. The first is the potential negative consequences felt by junior (non-tenured) scientists or minorities, who could, at least in theory, face severe repercussions for criticizing the work of senior/powerful colleagues who sit on editorial boards or grant panels. In my mind this conflict has never been properly addressed; while I believe it is completely reasonable for tenured scientists to be open and accountable in their reviews (I always sign mine), it is questionable whether this should be required uniformly across science. The second concern with open reviewing is the potential legal backlash of scientists being sued by litigious authors who feel aggrieved by a reviewer's published comments. We are seeing this already on PubPeer and elsewhere. This raises the question of who assumes legal responsibility for the content of a signed review. In the case of F1000Research, for instance, does it or will it provide legal indemnity to reviewers who choose to unmask themselves? Finally, I would prompt the authors of this paper to consider, and ideally speculate on, ways their oath (or code of practice as I would call it) might be implemented and incentivised in science. Could it, for instance, be worked into the next REF in some way? How could this be achieved and what challenges would need to be overcome? How does this initiative relate to other emerging group-led initiatives, such as the Agenda for Open Research (https://agendaforopenresearch.org/)? There authors have no shortage of good intentions but as we know, there lies a world of groupthink and social inertia between good intentions and good practices.Regardless of the above concerns, I applaud this much-needed call for greater transparency in the peer review process.", "responses": [ { "c_id": "1167", "date": "09 Jan 2015", "name": "Michael Markie", "role": "Author Response", "response": "Thanks Chris for your very helpful and candid report. Like all of the reviewers have suggested we have simplified the oath and made it much clearer and simpler to use. Yes, we agree that some of the language used was difficult for non-native English speakers and hence we have made it clearer to read in places that may of caused confusion. We also agree with your point about new/junior reviewers who may feel exposed by openly reviewing someone more senior than them and have acknowledged that in the guidelines. Here at F1000Research, we have implemented a system of co-authoring referee reports to help support junior researchers who feel that on their own they may be subject to repercussions from the authors. With regards to the legality of an open review on F1000Research, reviewers submit their report according to our terms and conditions which clearly state the report will be published under a CC BY license, and that the report must not be defamatory. However as a publisher we are also conscious about each review that is published on the site and we have system in place to ensure that nothing libel would be intentionally published. More generally, openness and transparency in signing a report means that they are on the whole much more civil and are of a constructive nature that encourages dialogue. We believe the peer review process should be a collaborative process and hence that is the environment we try to provide. Here is a letter of response from Rebecca Lawrence, Managing Director of F1000Research, recently published in response to the recent libel story from the anonymous review on PubPeer: http://www.timeshighereducation.co.uk/comment/letters/an-anonymity-problem/2017051.article. Finally, just after we published the review we became aware of the Agenda for Open Research who have similar goals that align with us and they were kind enough to link to the oath in their guideline for reviewers. We fully support their initiative, and we will seek opportunities to collaborate where possible. You are right, it would be great to push to get open reviewing to be recognised as an official part of the REF (and other funding bodies) and acknowledged as a measurable scientific output. Here at F1000Research we are taking steps to make this happen; currently we mint each report with a DOI and we are heavily involved in the Peer Review Service project in collaboration with ORCID and CASRAI." } ] }, { "id": "6802", "date": "27 Nov 2014", "name": "Etienne Joly", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript was written by participants of a workshop entitled \"AllBio: Open Science & Reproducibility Best Practice Workshop\" which took place at the TGAC in Norwich UK in September 2014. The alleged purpose of the oath and manifesto proposed in this manuscript is to make \"transparency, reproducibility and citizen-scientist engagement the default parameters for performing sound research.\"Those goals are clearly highly laudable, and I completely agree that the process of scientific refereeing would greatly improve by going 'open'. I do, however, feel very ill at ease about acting as a referee for this particular paper, for the following three main reasons:This is not a scientific manuscript, and it neither contains data, nor reviews a scientific topic. I am thus left wondering why such a manuscript should need to be peer reviewed. After reading it, I certainly cannot conclude that it is scientifically sound. The best I can conclude is that it is not scientifically unsound. Although this manuscript is very short, I must say that I found it rather difficult to read because of its structure which contains many redundancies and of the fuzziness of its purpose. One aspect that particularly bothers me is the unquestioned assumption that open refereeing will improve reproducibility. The first thing the authors should clarify is what they mean by reproducibility since there are at least two types that I can think of:The first one concerns data reproducibility (or robustness). In other words will similar data, leading to the same conclusions, be obtained if the experiment is repeated (on a different day and/or with different samples and/or in a different place and/or by different people etc…). The second type of reproducibility relates to the capacity of other scientists to reproduce an experiment described in a published manuscript. I suspect that when they refer to 'science reproducibility', the authors refer to the latter type, although the first type is the most important one in my eyes. The authors duly acknowledge that they have been inspired by reviewer's oaths previously proposed by others (refs 13-15), and if I am being completely honest, I do not find that the set of 17 rules they propose represent a significant improvement on those previously proposed oaths. Because I do not want to plagiarise Jonathan Eisen, I will simply suggest that the authors should very seriously consider following the many suggestions he has made on his blog to improve this paper (http://icis.ucdavis.edu/?p=505 ), and especially the idea of reversing the Oath and the Manifesto.", "responses": [ { "c_id": "1172", "date": "12 Jan 2015", "name": "Dan MacLean", "role": "Author Response", "response": "Thanks Etienne for your very helpful and candid report. We agree that the article needed to be made clearer in order to convey its intentions to the reader and we have taken measures to do this by clearly elucidating the open science principle. We have also made it clear about what we mean by reproducibility, and how the robustness of research should mean that similar data should be able to produce the same conclusions. We have also simplified the oath on the advice given to us from you and the other reviewers to make it easier to read and reuse." } ] }, { "id": "6987", "date": "09 Dec 2014", "name": "Suzanne Scarlata", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI think this article makes many good points, and I also agree with the other critiques. However, I do see danger in full transparency. The problem is that significance of study can be subjective and used in a biased way to sway readers towards a view of greater importance, in order to achieve a good standing with the authors. At this point in time, it is important for editors of other non-transparent journals to remind reviewers of the key aspects of this oath – to critically read the paper and make constructive comments as best as they can. Editors need to use their power to delete inappropriate reviews or reviewers.", "responses": [ { "c_id": "1170", "date": "12 Jan 2015", "name": "Dan MacLean", "role": "Author Response", "response": "Thanks Suzanne for your helpful and candid report. We have now satisfied the other reviews to make the oath clearer and simpler to use. We take your point that perceived impact can indeed sway readers and we certainly agree that as well as the scientific community practicing open science and reviewing openly, journals and journal editors need to play a role in ensuring that reviewers are doing a job that ensures integrity - we believe they can use the principles of the oath to do this." } ] }, { "id": "6800", "date": "10 Dec 2014", "name": "Lawrence Patrick Kane", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe principles outlined here are important, and this piece is certainly timely. I have two suggestions to improve the manuscript. First, as currently constituted, the manuscript is a bit repetitive, with a large text box recapitulating what is also laid out in much the same format in the body of the manuscript. Perhaps each of the major points could be expounded upon in the text. Second, while it is hard to argue with the specific tenets enumerated here, there are quite a lot of them. I guess my feeling is that for ideas like this to be more widely adopted that it would be helpful if the core principles could be boiled down to a more manageable size (which would be ideally presented in a text box). I applaud the authors for doing their part to increase openness in scientific publishing, something I agree is very much needed.", "responses": [ { "c_id": "1169", "date": "12 Jan 2015", "name": "Dan MacLean", "role": "Author Response", "response": "Thanks for your helpful and candid report Larry. We agree the manuscript was a little repetitive and so we have made it much more succinct, by concentrating on the open science principle we want to champion. We have also condensed the oath into 4 principles with an accompanying rationale and made it available on FigShare, which should make it much easier to follow and reuse." } ] } ]	1	https://f1000research.com/articles/3-271
https://f1000research.com/articles/4-3/v1	07 Jan 15	{ "type": "Correspondence", "title": "Further available immunization option to prevent pneumococcal disease", "authors": [ "Ivo Vojtek", "Bernard Hoet", "Bernard Hoet" ], "abstract": "In their recent review, Charles Feldman and Ronald Anderson provide an overview of various clinical aspects of pneumococcal infections. We would like to complete this report by providing some additional information on a widely-used immunization option, which was not originally mentioned in the article. The protein D pneumococcal conjugate vaccine (PHiD-CV) has been pre-approved by WHO and its impact is supported by real-life data from the regions of its use.", "keywords": [ "PHiD-CV", "efficacy", "post-marketing surveillance" ], "content": "Correspondence\n\nWe write in response to the report by Charles Feldman and Ronald Anderson about the recent advances in the understanding of Streptococcus pneumoniae infections1.\n\nWhile this article provided an informative and complete review of the current burden of the disease, pathogenesis and therapeutic options, we have noted a significant omission in the chapter dealing with available immunization strategies, which did not mention the WHO prequalified Pneumococcal Nontypeable Haemophilus influenzae Protein D Conjugate Vaccine (PHiD-CV; GSK Vaccines, Belgium). This vaccine is currently licensed in more than 125 countries with more than 200 million doses distributed as of August 2014 and is used in vaccination programmes in more than 40 countries or regions.\n\nWe feel it is important that health care professionals are made aware of the available evidence supporting the use of this vaccine in order that they are able to make an informed choice about the best care for their patients, and therefore we provide additional information to supplement the review article. It is the only modern pneumococcal conjugate vaccine with impact on invasive pneumococcal disease, pneumonia and acute otitis media that has been proven in two pivotal randomized controlled efficacy trials performed in Finland and Latin America2–4. Thanks to its world-wide use, there is also a plethora of post-marketing and epidemiology data spanning five continents, recently reviewed by Plosker8, that proves its impact on the pneumococcal disease and makes it a worth-while alternative to the pneumococcal conjugate vaccine PCV13 which the health care community should be made aware of5–7. We have summarized the main effectiveness and impact data in Table 1.\n\nIPD: Invasive Pneumococcal disease; VE: vaccine efficacy; RR: relative rate reduction.", "appendix": "Author contributions\n\n\n\nIV wrote the abstract and the main body of the article. BH supervised the process. Both authors critically edited the correspondence and agreed to the final content.\n\n\nCompeting interests\n\n\n\nIV and BH are employed by GSK group of companies. BH owns stock in GSK group of companies.\n\n\nGrant information\n\nGlaxoSmithKline Biologicals SA funded all costs associated with the development of this manuscript.\n\n\nAcknowledgments\n\nThe authors would like to thank Bram Blomme (XPE Pharma and Science c/o GSK) for editorial support.\n\n\nReferences\n\nFeldman C, Anderson R: Recent advances in our understanding of Streptococcus pneumoniae infection. F1000Prime Rep. 2014; 6: 82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPalmu AA, Jokinen J, Borys D, et al.: Effectiveness of the ten-valent pneumococcal Haemophilus influenzae protein D conjugate vaccine (PHiD-CV10) against invasive pneumococcal disease: a cluster randomised trial. Lancet. 2013; 381(9862): 214–22. PubMed Abstract \| Publisher Full Text\n\nTregnani MW, Saez-Llorens X, Lopez P, et al.: Efficacy of pneumococcal nontypable Haemophilus influenzae protein D conjugate vaccine (PHiD-CV) in young Latin American children: A double-blind randomized controlled trial. PLoS Med. 2014; 11(6): e1001657. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPalmu AA, Kilpi TM, Rinta-Kokko H, et al.: Impact of 10-Valent Pneumococcal Conjugate Vaccine (PCV10) against Hospital-Diagnosed Pneumonia among Vaccine-Eligible Children in Finland. Presented at: 54th Interscience Conference on Antimicrobial Agents and Chemotherapy; September 5–9, 2014, Washington DC. Reference Source\n\nDeceuninck G, De Wals P: Effectiveness of Three Pneumococcal Conjugate Vaccines (PCVs) to Prevent Invasive Pneumococcal Disease (IPD) in Quebec, Canada. Presented at: 9th International Symposium on Pneumococci and Pneumococcal Diseases; March 9–13, 2014, Hyderabad, India. Reference Source\n\nDomingues CM, Verani JR, Montenegro Renoiner EI, et al.: Effectiveness of ten-valent pneumococcal conjugate vaccine against invasive pneumococcal disease in Brazil: a matched case-control study. Lancet Respir Med. 2014; 2(6): 464–71. PubMed Abstract \| Publisher Full Text\n\nJokinen J, Rinta-Kokko H, Siira L, et al.: Impact of 10-valent pneumococcal conjugate vaccine (PCV10) on invasive pneumococcal disease (IPD) among vaccine-eligible children in Finland. Presented at: 8th World Congress of the World Society for Pediatric Infectious Diseases (WSPID), November 19–22, 2013, Capetown.\n\nPlosker GL: 10-Valent pneumococcal non-typeable Haemophilus influenzae protein D-conjugate vaccine: a review in infants and children. Paediatr Drugs. 2014; 16(5): 425–44. PubMed Abstract \| Publisher Full Text" }	[ { "id": "7220", "date": "20 Jan 2015", "name": "Paola Marchisio", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI read with great interest the letter of Vojtek and Hoet. Well done and necessary. I have some remarks and suggestions:In the abstract the term “pre-approved” is not clear to all the readers. The exact date of approval would be useful because it could inform the readers that PHiD-CV has a long and great history. Page 2: “prequalified” is not clear (see above) Page 2 , second column, third line. I would add “evidence based” before “informed choice” in order to stress the big amount of available rigorous data. Page 2, 11th line: I would say “proves its beneficial impact…” It should be underlined that PHiD-CV is approved for use in children younger than 5 years of age (that could be one the reason why it was not quoted in the paper of Feldam which focuses mostly on adult/elderly patients) In the table I would add “schedule” before “3+1” or “2+1” . In the current version one may read “minus 3 plus 1” and may misunderstand.", "responses": [] }, { "id": "7587", "date": "23 Mar 2015", "name": "Marco Safadi", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe correspondence article “Further available immunization option to prevent pneumococcal disease” from Vojtek & Hoet provides relevant information regarding one of the currently available pneumococcal conjugate vaccines. This information was missing in the review article (Recent advances in our understanding of Streptococcus pneumoniae infection ) written by Feldman & Anderson. The correspondence article is well written and should be considered for indexing.I have only minor comments:In the second paragraph, when mentioning that the PHiD-CV vaccine is currently licensed in more than 125 countries, the authors should make clear that the vaccine is licensed for active immunization against invasive disease, pneumonia, and acute otitis media (AOM) caused by S. pneumoniae in infants and young children up to 5 years of age. In the third paragraph, when mentioning that the PHiD-CV vaccine is a worth-while alternative to the pneumococcal conjugate vaccine PCV13, the authors should add: … in children younger than 5 years of age. In Table 1, when showing the different study results observed with the PHiD-CV vaccine, the authors should specify which results are vaccine efficacy data and which are vaccine effectiveness data.", "responses": [] } ]	1	https://f1000research.com/articles/4-3
https://f1000research.com/articles/4-2/v1	05 Jan 15	{ "type": "Observation Article", "title": "High reproductive synchrony of Acropora (Anthozoa: Scleractinia) in the Gulf of Aqaba, Red Sea", "authors": [ "Jessica Bouwmeester", "Michael L. Berumen", "Michael L. Berumen" ], "abstract": "Coral spawning in the northern Gulf of Aqaba has been reported to be asynchronous, making it almost unique when compared to other regions in the world. Here, we document the reproductive condition of Acropora corals in early June 2014 in Dahab, in the Gulf of Aqaba, 125 km south of previous studies conducted in Eilat, Israel. Seventy-eight percent of Acropora colonies from 14 species had mature eggs, indicating that most colonies will spawn on or around the June full moon, with a very high probability of multi-species synchronous spawning. Given the proximity to Eilat, we predict that a comparable sampling protocol would detect similar levels of reproductive synchrony throughout the Gulf of Aqaba consistent with the hypothesis that high levels of spawning synchrony are a feature of all speciose coral assemblages.", "keywords": [ "Multi-species synchronous spawning of scleractinian corals is a feature reported from almost all speciose coral assemblages (Baird et al.", "2009a", "Baird et al.", "2010", "Raj & Edwards", "2010)", "including the Arabian Sea (Baird et al.", "2014)", "and the Red Sea (Bouwmeester et al.", "2011", "Bouwmeester et al.", "2014", "Hanafy et al.", "2010). A notable exception is at Eilat", "on the Israeli coast", "in the Gulf of Aqaba", "where spawning is described as asynchronous with different species spawning in different seasons", "on different months", "and at different stages of the lunar cycle", "with no overlap in spawning between species (Shlesinger & Loya", "1985", "Shlesinger et al.", "1998)." ], "content": "Observation\n\nMulti-species synchronous spawning of scleractinian corals is a feature reported from almost all speciose coral assemblages (Baird et al., 2009a; Baird et al., 2010; Raj & Edwards, 2010), including the Arabian Sea (Baird et al., 2014), and the Red Sea (Bouwmeester et al., 2011; Bouwmeester et al., 2014; Hanafy et al., 2010). A notable exception is at Eilat, on the Israeli coast, in the Gulf of Aqaba, where spawning is described as asynchronous with different species spawning in different seasons, on different months, and at different stages of the lunar cycle, with no overlap in spawning between species (Shlesinger & Loya, 1985; Shlesinger et al., 1998).\n\nHere, we quantify the reproductive synchrony of Acropora corals in Dahab, on the Egyptian coast of the Gulf of Aqaba, 125 km south of Eilat, Israel (Figure 1). Among Red Sea reef habitats, fringing reefs in the Gulf of Aqaba support distinct coral assemblages with high cover and diversity of hard corals (DeVantier et al., 2000).\n\nMap of a the Red Sea, showing the location of previous work on scleractinian coral spawning in the Red Sea, and b the Gulf of Aqaba, showing Dahab, our study site.\n\nThe reproductive condition of 90 colonies from 15 Acropora species was assessed at two dive sites in Dahab, Um Sid (28° 25’14.16”N, 34° 27’27.52”E) and Eel Garden (28° 30’19.21”N, 34° 31’15.58”E), from the 2nd to the 4th of June 2014, a week before the full moon that month (Table 1). Acropora colonies at 1–10m depth were examined on snorkel by breaking 1–3 coral branches below the sterile apical zone to expose the developing oocytes. Colonies were recorded as mature when oocytes were visible and pigmented (Figure 2), immature when oocytes were visible and white, and empty when oocytes were too small to see with the naked eye or absent (following Baird et al., 2002). Colonies with mature oocytes are highly likely to spawn close to the night of the next full moon (in this case, the June full moon), whereas colonies with immature eggs are likely to spawn on or around a full moon one or two months later (in this case the July or August full moon). Colonies without oocytes have either already spawned or are unlikely to do so for at least three months.\n\nn: number of sampled colonies.\n\na Exposed oocytes in a mature colony of Acropora variolosa b close-up of pigmented oocytes.\n\nSeventy-one percent of Acropora colonies had mature oocytes and an additional three percent had immature oocytes (Table 1). No oocytes were observed in the remaining colonies. Fourteen out of 15 species had at least one colony with mature eggs, and in seven of those species, 100% of the sampled colonies had mature eggs (Table 1).\n\nThe reproductive condition in the Acropora assemblage at Dahab in June is very similar to that estimated in Acropora assemblages on the Egyptian coast of the northern Red Sea, where 85% of colonies from 12 species had mature oocytes in Marsa Alam in April 2008 and 99% of colonies from 17 species had mature oocytes in Hurghada in April 2009 (Hanafy et al., 2010). Subsequent sampling in both years revealed the absence of oocytes in all but one of these species, indicating that spawning had occurred sometime in the previous couple of weeks, most likely around the full moon of April (Hanafy et al., 2010). Nighttime observations in 2012 in Hurghada revealed spawning of 12 Acropora species over two consecutive nights around the full moon of May (Kotb, 2012). Similarly, 13 Acropora species in Thuwal, central Red Sea (Figure 1a), were observed to spawn together on the same night, both in April 2011 and in April 2012, following initial reproductive surveys which revealed 65% of mature Acropora colonies from 9 species in 2011 and 39% of mature Acropora colonies from 16 species in 2012 (Bouwmeester et al., 2014). The high percentage of species and colonies with mature oocytes in Dahab one week before the June full moon strongly suggests they will spawn synchronously as observed in Thuwal in the central Red Sea (Bouwmeester et al., 2011; Bouwmeester et al., 2014) and in Hurghada in the northern Red Sea (Hanafy et al., 2010; Kotb, 2012). Broadcast spawning of corals in most locations of the Indo-Pacific occurs as sea surface temperatures are increasing or when temperatures are close to their annual maxima (Baird et al., 2009a). In Dahab, waters start warming in the months of March-April, rising from 21–22°C to a maximum of 26–27°C in the month of August (Cornils et al., 2007; Plähn et al., 2002). Spawning in June most likely occurs when temperatures are ~24–25°C, possibly an optimum temperature for spawning and early larval development in the Gulf of Aqaba.\n\nThe month of spawning of Acropora species in the Gulf of Aqaba is two months later than in the northern and central Red Sea, where most Acropora spawn in April (Bouwmeester et al., 2014; Hanafy et al., 2010). This one or two-month offset is not surprising due to the difference in local temperature regimes and is similar to the latitudinal pattern observed along the east coast of Australia and from the Philippines to Japan (Baird et al., 2009b). Spawning in Dahab does not seem to occur before the waters reach 24–25°C, suggesting that a minimal temperature threshold is required during the warming of surface waters for spawning. In the central Red Sea, those temperatures are reached in March-April, and indeed multi-species spawning of Acropora has been recorded in April at 25–27°C (Bouwmeester et al., 2014).\n\nOur data from Dahab match the data from Eilat (Shlesinger & Loya, 1985) for the timing of Acropora spawning in the Gulf of Aqaba, however, the larger number of Acropora species examined in the present study allows us to understand reproductive synchrony within this genus much more effectively. Indeed, we predict that with a comparable sampling protocol, similar levels of Acropora reproductive synchrony would be detected at Eilat, only 125 km north of Dahab, and would support the hypothesis that high levels of spawning synchrony are a feature of all speciose coral assemblages (Guest et al., 2005). The length of the scleractinian reproductive season can be established by sampling distantly related species from the coral assemblage, which in Eilat lasts four months for broadcast spawning species (Guest et al., 2005; Shlesinger & Loya 1985; Shlesinger et al., 1998), but sampling closely related species such as Acropora species will determine whether overlap in spawning occurs and will allow estimation of the level of synchrony in the assemblage.", "appendix": "Author contributions\n\n\n\nJB conceived the study and collected the data. All authors wrote the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nFunding was provided by KAUST grants (URF/1/1389-01-01 and CCF/1/1973-01-01) as well as baseline research funds to MLB.\n\n\nAcknowledgements\n\nWe are grateful to Andrew H. Baird for providing constructive comments on the manuscript. We also thank Hagag, Hamd, Eid, Selim, and Barbara for logistical support in Dahab.\n\n\nReferences\n\nBaird AH, Marshall PA, Wolstenholme JK: Latitudinal variation in the reproduction of Acropora in the Coral Sea. Proc 9th Int Coral Reef Symp. 2002; 1: 385–389. Reference Source\n\nBaird AH, Guest JR, Willis BL: Systematic and biogeographical patterns in the reproductive biology of scleractinian corals. Annu Rev of Ecol Evol Syst. 2009a; 40: 551–571. Publisher Full Text\n\nBaird AH, Birrell CL, Hughes TP, et al.: Latitudinal variation in reproductive synchrony in Acropora assemblages: Japan vs. Australia. Galaxea. 2009b; 11(2): 101–108. Publisher Full Text\n\nBaird AH, Kospartov MC, Purcell S: Reproductive synchrony in Acropora assemblages on reefs of New Caledonia. Pac Sci. 2010; 64(3): 405–412. Publisher Full Text\n\nBaird AH, Abrego D, Howells EJ, et al.: The reproductive season of Acropora in Socotra, Yemen. [v2; ref status: indexed, http://f1000r.es/392] F1000Res. 2014; 3: 78. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBouwmeester J, Khalil MT, De La Torre P, et al.: Synchronous spawning of Acropora in the Red Sea. Coral Reefs. 2011; 30(4): 1011–1011. Publisher Full Text\n\nBouwmeester J, Baird AH, Chen CJ, et al.: Multi-species spawning synchrony within scleractinian coral assemblages in the Red Sea. Coral Reefs. 2014; 1–13. Publisher Full Text\n\nCornils A, Schnack-Schiel S, Al-Najjar T, et al.: The seasonal cycle of the epipelagic mesozooplankton in the northern Gulf of Aqaba (Red Sea). J Mar Syst. 2007; 68(1–2): 278–292. Publisher Full Text\n\nDeVantier L, Turak E, Al-Shaikh K, et al.: Coral communities of the central-northern Saudi Arabian Red Sea. Fauna of Arabia. 2000; 18: 23–66. Reference Source\n\nGuest JR, Baird AH, Goh BPL, et al.: Seasonal reproduction in equatorial reef corals. Inverteb Reprod Dev. 2005; 48: 207–218. Publisher Full Text\n\nHanafy MH, Aamer MA, Habib M, et al.: Synchronous reproduction of corals in the Red Sea. Coral Reefs. 2010; 29(1): 119–124. Publisher Full Text\n\nKotb MMA: Reef Check spotlight: Synchronized coral spawning in the Red Sea. The Transect Line. 2012; 2. Reference Source\n\nPlähn O, Baschek B, Badewien TH, et al.: Importance of the Gulf of Aqaba for the formation of bottom water in the Red Sea. J Geophys Res. 2002; 107(C8): 22-1–22-18. Publisher Full Text\n\nRaj KD, Patterson J: Observations on the reproduction of Acropora corals along the Tuticorin coast of the Gulf of Mannar, Southeastern India. Indian J Mar Sci. 2010; 39(2): 219–226. Reference Source\n\nShlesinger Y, Loya Y: Coral community reproductive patterns: Red Sea versus the Great Barrier Reef. Science. 1985; 228(4705): 1333–1335. PubMed Abstract \| Publisher Full Text\n\nShlesinger Y, Goulet T, Loya Y: Reproductive patterns of scleractinian corals in the northern Red Sea. Mar Biol. 1998; 132(4): 691–701. Publisher Full Text" }	[ { "id": "7207", "date": "07 Jan 2015", "name": "Jean Kenyon", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe research uses an accepted method of assessing reproductive condition in Acropora colonies. The article clearly and concisely reports the location, species, and number of colonies sampled and provides background context on other coral reproductive sampling in the Red Sea, which combine to bring the reader to the same conclusions as the authors regarding multi-species spawning synchrony in this region. One hopes the authors will find the resources to test their hypothesis that similar levels of Acropora reproductive synchrony would be detected at Eilat using a comparable sampling protocol. Overall, the writing (including title, abstract, and main body of text) is succinct and provides an appropriate level of detail.", "responses": [] }, { "id": "7208", "date": "16 Jan 2015", "name": "Andrew G. Bauman", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article presents information on the reproductive synchrony of 15 Acropora species in the southern Gulf of Aqaba, Red Sea. This is a sound study that used well-established sampling techniques, and provides adequate details for the methods, results and findings. Their results showed a high proportion of Acropora colonies (78%) sampled prior to the full moon in June had mature oocytes and suggest that most colonies will spawn in June, with a high probability of multi-species spawning. Most interestingly, their data matched previously spawning records from Eilat, 125km north of their site, providing additional evidence that high levels of spawning synchrony are likely a feature of all speciose coral assemblages. NOTE: While I agree with the authors that it is likely that the colonies with mature (pigmented) oocytes are generally considered to spawn on, or shortly after, the subsequent full moon, conducting follow up surveys in the subsequent months (i.e., July and August) would confirm whether colonies are in fact releasing all of their gametes. In a similar reproductive study conducted in the neighboring Persian Gulf (Bauman et al., 2011) mature colonies were found over four consecutive months suggesting that some colonies are either not releasing mature gametes or that some colonies are spawning twice.", "responses": [] } ]	1	https://f1000research.com/articles/4-2
https://f1000research.com/articles/3-82/v1	02 Apr 14	{ "type": "Research Article", "title": "Working memory training shows immediate and long-term effects on cognitive performance in children and adolescents", "authors": [ "Fiona Pugin", "Andreas J. Metz", "Madlaina Stauffer", "Martin Wolf", "Oskar G. Jenni", "Reto Huber", "Fiona Pugin", "Andreas J. Metz", "Madlaina Stauffer", "Martin Wolf", "Reto Huber" ], "abstract": "Working memory is important for mental reasoning and learning processes. Several studies in adults and school-age children have shown performance improvement in cognitive tests after working memory training. Our aim was to examine not only immediate but also long-term effects of intensive working memory training on cognitive performance tests in children and adolescents. Fourteen healthy male subjects between 10 and 16 years trained a visuospatial n-back task over 3 weeks (30 min daily), while 15 individuals of the same age range served as a passive control group. Significant differences in immediate (after 3 weeks of training) and long-term effects (after 2-6 months) in an auditory n-back task were observed compared to controls (2.5 fold immediate and 4.7 fold long-term increase in the training group compared to the controls). The improvement was more pronounced in subjects who improved their performance during the training. Other cognitive functions (matrices test and Stroop task) did not change when comparing the training group to the control group. We conclude that spatial working memory training in children and adolescents boosts performance in similar memory tasks such as the auditory n-back task. The sustained performance improvement several months after the training supports the effectiveness of the training.", "keywords": [ "Learning is crucial for adaptation to new situations and is essential for improvements in cognitive functions over time. One important aspect of learning-related improvements in cognitive functions is working memory", "which is the ability of simultaneously store and process information held in mind despite irrelevant", "potentially interfering stimuli1. It is generally accepted that working memory processes support higher cognitive functions including reasoning1." ], "content": "Introduction\n\nLearning is crucial for adaptation to new situations and is essential for improvements in cognitive functions over time. One important aspect of learning-related improvements in cognitive functions is working memory, which is the ability of simultaneously store and process information held in mind despite irrelevant, potentially interfering stimuli1. It is generally accepted that working memory processes support higher cognitive functions including reasoning1.\n\nSeveral studies have assessed working memory capacity during childhood and around the age of school entry, at a time when the learning load is large. For example, Alloway et al. showed that working memory impairment seems to be disadvantageous for learning abilities in reading and mathematics2. In addition, using the Conners’ Teacher Rating Scale, school teachers have reported high inattentiveness and executive problems in those children with poor working memory2.\n\nRegarding the predictive power of working memory for learning processes, it is not surprising that difficulties in cognitive functions such as reading3 and mathematics4 are associated with working memory skills. For example, Wang et al. showed that children between 8 and 10 years with difficulties in single word reading performed worse in simple and complex span tasks compared to controls, two widely used working memory tasks3. In another study, mathematical learning disabilities of school-age children were shown to be associated with low performance in spatial working memory4.\n\nConsidering the impact of working memory on a wide range of cognitive functions, the enhancement of working memory is of great interest. Studies in adults showed that working memory may be trained in elderly people of around 80 years5 as well as in young adults in their twenties6. Also in pre-school children, visuospatial working memory training improved untrained verbal working memory performance7. Some of these studies not only showed significant performance increases in the trained working memory tasks, but also in other, untrained working memory tasks5 and functionally more distant tasks such as fluid intelligence tests (transfer effect)6.\n\nIn a more recent study involving school-age children of around 9 years, Jaeggi et al. also investigated the long-term effects of working memory training8. Immediately after the training, they observed significant higher fluid intelligence in the group with a large training gain compared to the small gain group or a control group. Thus, the larger the training improvement, the greater were the transfer effects. Interestingly, the gain in fluid intelligence remained stable after three months without additional training in the large training gain group8.\n\nThe aim of our study was to investigate working memory training and its effects on working memory tasks and fluid intelligence in male subjects between 10 and 16 years. In fact, this age range may be particular susceptible to interventions because many cognitive functions are still developing9. Furthermore, working memory performance has been shown to be linked to attentional control10 and processing speed11. Thus, putative transfer effects on fluid intelligence may not be limited to fluid intelligence, but may also include other cognitive functions. Hence, in addition to working memory and fluid intelligence tasks, we also measured inhibition and interference tasks as well as standardized processing speed. Finally, long-term effects may be indicative for the effectiveness of the training, thus, cognitive testing was repeated not only immediately after the training period but also a few months later.\n\n\nMaterials and methods\n\nThe participants were recruited through print media and announcements (e.g., community centers, sport clubs, schools). Inclusion criteria (evaluated by phone screening questionnaires) were: male, age between 10 to 16 years, good general health, right-handedness, no neurological disorder or other disease, no learning disabilities, no smoking or heavy alcohol or caffeine consumption (more than one serving per week). Parents and their children gave written informed consent after explanation of the study methods and aims. Furthermore, ethical clearance was given by the local institutional review board (IRB, KEK-ZH-Nr. 2010-0238/2). In order to assign the subjects to the experimental and control group, we used a stratified randomization with age and cognitive performance in matrices test and letter-number-sequencing (MAT and LNS, see below) as stratification factor.\n\nWe assessed the cognitive performance before (PRE) and after 3 weeks (POST) of training. In addition, all subjects were asked to participate in a third session after a minimum of 2 months (FOLLOW-UP = FU, Figure 1). In one subject (control subject, code 36), POST took place 1 week later due to sickness on the planned test date.\n\nCognitive testing included two working memory tasks (auditory n-back and letter-number sequencing), a fluid intelligence task [matrix reasoning task, TONI-IV (Test of Non-verbal Intelligence Version IV)], two cognitive control tasks (Stroop and Flanker task), two processing speed tasks (symbol search and digit-symbol substitution task) and a short-term memory task (number-span task). In addition, subjective motivation and concentration were measured on a scale from 1 (minimal) to 10 (maximal).\n\nWithin the 3 weeks between PRE and POST, 14 of the subjects completed an intensive working memory training programme (training group), which consisted of an adaptive visuospatial n-back task12. The passive control group (N = 15) did not receive any working memory training.\n\nThe subjects were unaware of their group affiliation until the end of the first cognitive test session (PRE). The day after PRE testing, each subject from the training group was introduced to the training task by the research assistant. By means of an information sheet, the participants were informed in detail about the task and the setting, and completed a self-motivational control sheet. After this first supervised training session, the participants were able to independently perform the training sessions. Thereafter, the participants were asked to train at home for a maximum of 30 minutes per day over the following 3 weeks. Within these 20 days, they were visited once at home at a planned date by a research assistant (not the cognitive test examiner). During this visit, the working memory training compliance was evaluated. For two subjects, the home visit did not take place due to organizational reasons (control group subjects code 15 and 28). Three weeks after PRE testing, the cognitive testing was repeated (POST) and some months later, the subjects participated in the third test session (FU, range 2 months 22 days to 5 months 6 days). No difference in the timing of FU was observed between the groups (training group: 3 months 21 days ± 6.44 mean ± SEM; control group: 3 months 16 days ± 4.77 mean ± SEM, unpaired t-test).\n\nAll participants received a present for taking part in the 3 cognitive test sessions. For participating in the 3 weeks of training, the members of the training group were given a small present of choice.\n\nCognitive performance of all but one subject was assessed by the same examiner. For one subject (code 28), the examiner was different at PRE due to organizational limitations. For each subject, the place of cognitive testing, time of day and week day were kept constant for all three sessions. Due to several reasons (e.g., time limitations or due to participant’s lack of motivation), the number of subjects was unequal for the cognitive tests (Table 1).\n\nPRE = session 1; POST = session 2, three weeks after session 1; FU = follow-up after 15.59 ± 0.56 weeks (mean ± SEM) after POST. Statistics: mixed ANOVA with factors ‘test session’ (PRE, POST, FU) and ‘group’ (training, control) for each measure. * p < 0.05, () < 0.1; a = age as covariate. Effect size: η2 square; RT = reaction time.\n\nVisual n-back training [VNBT, computerized version, BrainTwister software,12]. One important aspect of working memory training is the adaptation of the difficulty level to the subject’s performance. The working memory load should always be maximal13. For each stimulus of 20 + n consecutive stimuli in a series, the participants had to remember the position of a blue square on a black computer screen and indicate by button-pressing when the square was in the same position as n before. No response was afforded if the square was not in the same position as n before. During the test, the participants were supposed to fixate on a cross in the middle of the screen. Per trial, there were 8 possible positions (randomized) of the square (stimulus). Over 20 days, the participants were requested to train for 30 minutes per day. Each of the training sessions started with n = 2 and included several runs (each with a series of 20 + n). After each run, the feedback on performance in percent was displayed (only wrong trial clicks were included in the performance evaluation). The n increased if the performance level was over 90% or decreased with a performance level of 70% or less12.\n\nAuditory n-back task (ANB, computerized version, BrainTwister software). The auditory n-back task12 was based on the same underlying principle as the visuospatial training task, but with computerized spoken letters (C, I, K, Q, W, etc.) instead of visual squares. The duration of the task was restricted to 10 minutes. The maximal n of PRE, POST and FU were used as the dependent variable.\n\nLetter-number sequencing task (LNS, German version of the WISC-IV). The letter-number sequencing task was used as an additional task to assess working memory performance14. In this task, the examiner orally presented three times the same length of span (sequence), including a mixture of letters and numbers (e.g., 1 - B - 3 - G - 7). The subject had to remember the span and recite first the numbers in ascending order, then the letters in alphabetical order. As soon as the subject gave an incorrect answer for all three trials for a certain span, the task was finished. The number of the last correct span served as dependent variable (age standardized values according to the normative sample presented in the manual).\n\nMatrix reasoning task [MAT, Test of Nonverbal Intelligence (TONI IV)]. As a measure of fluid intelligence, we used the matrix reasoning task TONI-IV15 including two versions, from which the order (A, B, A or B, A, B) was balanced between the groups (age standardized values available). In the task, the participants had to choose the only pattern that completed the matrix presented from a given sample of patterns. The tasks stopped when three of five consecutive trials were not correctly solved or if the maximal trial number was reached (60, none of the subjects reached the maximum).\n\nStroop task (ST, paper version). For a measure of inhibition, thus inhibiting a response to a distractor stimulus in presence of a target stimulus, we employed a paper version of the Stroop task (one version and no age standardized values available) including three paradigms: 1) reading written color names, 2) naming the colors of lines and 3) naming color-words (incongruent paradigm), where the written color name is different from the ink color of the written word. The participants had to name the ink color of the word as fast as possible, thereby inhibiting to read out the written (semantic) color name. The duration (in seconds) and the errors per paradigm were assessed. As a measure of inhibition, the median time for the incongruent paradigm was used in our study16.\n\nFlanker task (FT, computerized version). As another measure of inhibition, a computerized version of the Flanker task was used. The task was programmed with Presentation 14.8 (Neurobehavioral Systems) according to Stins et al.17. In this task, stimulus-response interference is induced by the flanking arrows (distractors) around a middle arrow (target stimulus). Specifically, the participants had to indicate by button pressing (keyboard letter ‘a’ for left and ‘l’ for right) the pointing direction of the middle arrow of the randomly presented trials (condition neutral: < or >, condition congruent: <<<<< or >>>>>, condition incongruent: <<><< or >><>>). The difference between the reaction times for incongruent and congruent trials served as dependent variable.\n\nSymbol search task and digit symbol substitution task (SST, DSS, German version of the WISC-IV). Processing speed was assessed with symbol search and digit symbol substitution task14. These tasks demand for quick and accurate responses, thereby challenging the ability of speed, accuracy and attention.\n\nIn each trial of the symbol search task, a target symbol was compared with a group of diverse symbols, and the participant indicated with YES or NO whether the chosen symbol was part of the presented group. In the second processing speed task, the DSS14, the participant had to assign a series of numbers to simple geometric symbols, according to a given key. Both tasks had to be solved as quickly as possible. The dependent variable was the number of correct trials within a certain time range (age standardized values).\n\nNumber-span task (NST, KAI). The number-span task, assessed with the KAI18, measures short-term memory. In this task, the participants had to repeat a span of numbers that was spoken by the examiner with a regular rhythm of one second. With every correct repetition, the length of the span increased, until the subject gave a false response, resulting in a following span with the same length. With a second error, the task was stopped. The maximally reached span length and the number of errors served as dependent variable (two versions, no age standardized values available).\n\nSubjective motivation and concentration. Before each cognitive test session, the participant was asked to rate his motivation and concentration on a scale (1 = not at all to 10 = highly motivated or concentrated).\n\nThe training performance was calculated from the individual mean n (n indicates the level of n-back performance, see VNB and ANB above) of each training session. Each session started at n = 2, independent of performance on the previous session. Thus, we decided to consider the first three runs of each session as adaptation runs and excluded them from the analysis. For each training participant, their maximal performance and their respective session were determined over the entire training period. The difference between maximal performance and the last training session (last level) and the level at the first training session (start level) were used as a measure for training gain. The training amount was defined as the number of runs.\n\nStatistical analysis was performed with SPSS software (PASW Statistics 18). The normal distribution of the cognitive variables was tested with Kolmogorov-Smirnoff test. The effects of the training on cognitive test performance were calculated with a mixed ANOVA, including factor ‘group’ (training, control) and factor ‘test’ (PRE, POST, FU), for each cognitive test. For tests with no age standardized values, age served as a covariate. Unpaired and paired t-tests were calculated for between-group respectively within-group effects. Due to the low number of participants, statistical trends were not considered.\n\n\nResults\n\nIn a first step, we analyzed the visuospatial n-back (VNB) training amount and improvement by comparing performances at the first session with the performances at the last session and with the individual maximal performances (Figure 2). Participants trained on average for 16.1 ± 1.2 days (mean ± SEM, range: 7 to 20 days). Training performance was significantly increased from the first to the last training session (1.48 ± 0.46 mean ± SEM, range -0.89 to 5.24, paired t-test, p < 0.05). Maximal performance was reached on average between session 10 and 11, at 66.9 % ± 7.4 (mean ± SEM, Figure 2) of the individual training time, and was significantly higher than performance at the end of the training (2.74 ± 0.49 mean ± SEM, range 0.66 to 5.65, paired t-test, p < 0.001 (Figure 2).\n\nIndividual training performance (first session, session of maximal performance, last session) is shown. Each solid line represents the performance of an individual (N = 14) in the visuospatial n-back (VNB) task training mean n of VBN at the first training session (circle), at the session of maximal performance (triangle) and at the last training session (square). The dashed line represents the average performance at the first session, the session of maximal performance and the performance at the last session. Average maximal performance was reached between session 10 and 11 (mean 10.21 ± SEM 1.22) and average performance at the last session was reached between session 16 and 17 (16.14 ± 1.19). * indicates: performance at session of maximal performance was significantly higher than performance at the first and the last training session. * indicates: performance at the last session was significantly higher than at the first session.\n\nTo assess the effect of age on training performance, we performed a correlational analysis. Performance during the first session was positively correlated with age (Figure 3, Pearson correlation, r = 0.76, p < 0.05), that is the older the child, the higher the initial performance. Gain or amount of training, however, was not correlated with age. Initial performance also positively correlated with maximal performance during the training (partial correlation with age as covariate, r = 0.59, p < 0.05).\n\nCorrelation between age (years) and performance at the first session of visuospatial n-back training (Pearson correlation, r = 0.76, p < 0.05).\n\nIn a next step, we analyzed performance in each cognitive test at PRE, POST and FU, comparing the training with the control group (Table 1). A mixed ANOVA test revealed a significant difference between ‘group’ and ‘test session’ in auditory n-back (ANB) performance (Table 1). No other test (letter-number sequencing task, number-span task, matrix reasoning task, Stroop task, and Flanker task) showed a significant change. Between-group analysis of performance differences showed significantly higher increases in maximal ANB performance from PRE to POST and to FU in the training compared to controls (Figure 4). The number of days between PRE and FU (i.e., the time interval between the first and the last session) did not correlate with the improvements in ANB (Pearson correlation).\n\nThe training group showed a significant increase from PRE to POST and to FU. The control group showed a significant increase from PRE to POST, but not FU. * indicates significant changes within group (training (red), control (black); paired t-test, p < 0.05. # indicates significant performance difference at the respective test session (unpaired t-test, p < 0.05).\n\nIn a following step, we analyzed the improvements in ANB in relation to the training gain and amount. Training gain, measured by the difference in performance between the first session and the session of maximal performance (Pearson correlation, r = 0.76, p < 0.05, Figure 5) as well as the difference between the first and the last session (Pearson correlation, r = 0.77, p < 0.05), correlated positively with the improvements from PRE to POST in ANB. No correlation between ANB increase and training amount was found.\n\nCorrelation of training gain (diffMx, difference between Maximal Performance and performance at the first training session) with the change in ANB from PRE to POST (r = 0.76, p < 0.05).\n\nFor assessing how training performance gain may influence the gain in auditory n-back (ANB), we grouped the subjects according to their training performance, by correlating the training performance over time with the session number (Figure 6). In seven of the 14 training subjects, training days positively correlated with daily performance, indicating a steady increase of training performance over the entire training period (steady performer group). In the remaining subjects, the training performance was not stable and/or decreased (unsteady performer group).\n\nSteady performers: individuals with a significant positive correlation between mean training performance (mean n) per session and the training session. Unsteady performers: no positive correlation. Left: mean ± SEM of n [visuospatial training task (VNB)] over each training session per group. Right: individual mean n (VNB) per training session. Blue: steady performer group. Green: unsteady performer group. # indicates significant performance differences between steady and unsteady performers at the first and the last session (unpaired t-test, p < 0.05).\n\nIn addition to the steady increase, the steady performers started on a significantly higher level (Figure 6) and showed a larger increase in training performance from the first session to the session of maximal performance and to the performance at the last session (unpaired t-test, p < 0.05).\n\nWhen we then compared ANB performance between these two groups, we found that the steady performers showed a significant higher increase from PRE to POST and higher increase in maximal ANB performance at POST and FU compared to the unsteady performers (Figure 7).\n\n* indicates significant changes within groups [steady performers (blue), unsteady performers (green)]. # indicates p < 0.05 (unpaired t-test between sessions [PRE, POST, FU]).\n\n\nDiscussion\n\nDuring three weeks of visuospatial memory training, the participants in this study showed a significant performance increase in auditory working memory compared to passive controls. The better performance remained high after some months. The performance improvement correlated with the training quality, that is the performance level, rather than with the amount of training. The cognitive control, measured with the Stroop and the Flanker task, was not associated with the performance increase. No significant transfer effects on fluid intelligence were observed.\n\nThe strongest effect of VNB training was found on ANB, a working memory task closely related to the one trained for by our participants. Importantly, not the amount of training but the training gain correlated positively with the immediate and long-term increase in the auditory working memory task: the higher the increase in ANB from PRE to POST and to FU, the higher the training gain. Our approach based on correlating the individual training sessions with the training performance at each session supports the finding that training performance rather than the amount of training is crucial for the increase in ANB. The correlations show that the participants with a steady increase of their training performance had larger gains in training performance as well as in immediate and long-term ANB performances.\n\nIn agreement with our results, few other studies reported long-term effects of working memory training on the working memory performance19,20. In young and old adults, n-back task performance was maintained three months after spatial working memory training20. Dahlin et al. found stable improvements even eighteen months after the training19. One explanation for such long-term effects in auditory working memory after training might be that training leads to more efficient use of working memory in daily life not only during the sessions, but also afterwards. A relationship between the efficient use of working memory and long term benefits for cognitive performance is supported by the observation that working memory tasks such as the digit span task and the visual-spatial working memory task were shown to predict mathematical and reading skills achieved in the first school years21. The transfer from test setting to daily life would be a convincing rationale to apply working memory training as a therapeutic tool. Indeed, in children with poor working memory performance, Holmes et al. found significant performance increases in mathematics 6 months after an intensive working memory training22. Thus, long-term measures of cognitive performance may indicate a certain effectiveness of cognitive training. If intensive working memory training is effective, a more efficient use of working memory would transfer into daily life, for example resulting in higher grades at school.\n\nThe idea that the observed improvements in visuospatial and auditory working memory would have occurred due to a use-dependent general increase in working memory capacity, may still be questioned by our finding that the letter-number sequencing, a typical task assessing working memory capacity, was not affected by training. This finding is in line with other studies comparing working memory capacity (e.g., by digit span tasks) and n-back tasks6,23. As Kane et al. suggested23, n-back tasks may ‘not reflect primarily a single construct’ because ‘complex span tasks typically demand serial recall’, whereas ‘n-back tasks typically demand recognition’. Thus, serial recall and recognition do not correlate. This observation may indicate that the auditory and visuospatial tasks are actually too similar to draw conclusions about general effects on working memory per se.\n\nWe found no significant effects of the training on non-verbal matrices test when comparing the training group to the control group. Also Jaeggi et al., when comparing fluid intelligence test performance between the experimental and control group and between three test sessions, did not find any effect of visuospatial working memory training on fluid intelligence8. Only after grouping the participants according to their training gain in a large and small training gain group (median split) some differences in transfer effects were observed: immediate transfer effects and, to lesser extent, long-term transfer effects were significantly higher in the large training gain group compared to the low training gain and control group. When we performed the same median split in our training group, we were not able to find any statistically significant transfer effects. However, several aspects, such as the training duration and the attractiveness of the training, render it difficult to directly compare the two studies. After all, in a study with adult participants, the transfer effects of working memory training on fluid intelligence could not be replicated24. Furthermore, in a recently presented meta-analysis that investigated the effects of working memory training on cognitive tasks, Melby-Lervåg and Hulme revealed that working memory training indeed improves the working memory capacity immediately after the training. The training, however, does not lead to transfer effects on fluid intelligence when focusing on studies with control groups and randomization25. Thus, despite the low number of participants included in this study and the lack of an active control group, our negative finding concerning transfer effects on fluid intelligence is congruent with the current literature. In fact, the passive control group had a very low bar to pass, and still no far-transfer effects were found. Thus, this finding may reflect strong evidence against far transfer effects.\n\nAs proposed by Diamond and Lee26, working memory training does not improve inhibition or processing speed. Our data are in line with this notion, since we did not find any effects of the training on the processing speed tasks and the Stroop and Flanker test. The training and the control groups increased processing speed over the three sessions similarly. As shown by Conway et al., digit-symbol tasks may rather reflect short-term memory than working memory27. However, our short-term memory task was not affected by working memory training. Thus, we conclude that processing speed, used for measuring digit-symbol and symbol search tasks, are unaffected by working memory training and/or practice effects may be stronger.\n\nOur data did not indicate that cognitive control, measured with the Stroop and Flanker task, is associated with the training gain and effects on auditory working memory. Although our results do not corroborate the theory of attentional control being a crucial factor for working memory28, we do not question the necessity of cognitive control for reasoning. Again, with our data sample of limited size and large inter-individual variability we may not have been able to capture factors that influence working memory performance gains or even changes in cognitive functions due to training.\n\nWe conclude from these results that the performance increase in a visuospatial working memory task is beneficial for auditory working memory in children and adolescents. The dominant long-term effects underline the importance of assessing performance not only right after the cognitive training, but also several months later. Regarding the importance of working memory for other cognitive functions, studies comparing populations of different developmental stage or of low and high working memory capacity are needed.\n\n\nData availability\n\nThe ethical approval granted to the authors by the IRB does not allow the publication of the raw data online. If readers would like to re-analyze the data set (for different purposes), additional ethical approval (on a individual user and purpose basis) will be required. The authors would be happy to support additional ethical approval applications from researchers for access to this data set.\n\n\nConsent\n\nInformed written consent was provided by the children participating in the study and their parents.", "appendix": "Author contributions\n\n\n\nMW, OGJ and RH conceived the study. Together with FP and MS, they designed the study. Recruitment of participants and data assessment were done by FP, AM and MS. Analysis was performed by FP and MS. FP, RH and OGJ wrote the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the University Research Priority Program “Integrative Human Physiology”, University of Zurich, Switzerland to MW, OGJ and RH; the Anna Müller Grocholsky foundation, Switzerland to OGJ and RH; the Clinical Research Priority Program “Sleep and Health, University of Zurich, Switzerland to RH; and the Swiss National Science Foundation grant P00P3-135438 to RH.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors thank Urs Bachofner for his support with data collection.\n\n\nReferences\n\nEngle RW: Working Memory Capacity as Executive Attention. Curr Dir Psychol Sci. 2002; 11(1): 19–23. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "5128", "date": "02 Jul 2014", "name": "Silvia A Bunge", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study examines the immediate and long-term cognitive effects of a 20-day visuospatial working memory training program in a small group of 10-16-year-old boys (N=14), in comparison with a passive control group (N=15). The intervention group showed improvements in visuospatial n-back task (training task) and an auditory n-back task (transfer task) that were significantly greater than those observed for the passive control group. Gains in the auditory n-back task were sustained at a three-month follow-up assessment. Measures of fluid intelligence, inhibition, processing speed, and other working memory tasks (letter-number sequencing task) did not show transfer effects at any time point, as judged by significance testing. The authors show differences in the degree of improvement on the transfer task as a function of training gains on the n-back task (steady vs. unsteady performers). This study adds to a growing body of work establishing the boundary conditions of cognitive training effects. Limitations of this study include a small sample size, the absence of an active control group, and the failure to report on the motivation ratings gathered at each session, which could help to clarify differences between the steady and unsteady performers.", "responses": [ { "c_id": "1081", "date": "14 Nov 2014", "name": "Fiona Pugin", "role": "Author Response", "response": "Dear Professor Bunge, we would like to thank you for your helpful comments on the manuscript. We have addressed all of them in our point by point reply below.Limitations of this study include a small sample size, the absence of an active control group,We changed the wording in the ‘Discussion‘ section:“The major limitations of this study are the low number of subjects and the lack of an active control group. However, despite these limitations, our negative finding is […]” and the failure to report on the motivation ratings gathered at each session, which could help to clarify differences between the steady and unsteady performers.This is a similar question as asked by reviewer Prof. van der Zee: see for point 3 in the response to Prof. van der Zee." } ] }, { "id": "4725", "date": "02 Sep 2014", "name": "Eddy van der Zee", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis work shows the immediate and long-term effects of working memory training on cognitive performance of children (age 10-16). It is an interesting finding that 30 min of daily visuo-spatial working memory training can have sustained improvement in an auditory n-back task. Although the results seem to be sound despite the relative low group sizes and the lack of an active control group, some issues need further attention:It is not made clear enough what the passive control group is doing (or was instructed NOT to do) and particularly how this was controlled for. Although Table 1 is of help, the memory training compliance was said to be evaluated by a research assistant. Can more information on this evaluation be provided to convince the reader that it is likely that the compliance of the children was indeed as planned? Can more be said about the cause of the individual differences in training performance (the steady and unsteady performer groups)? Is it anticipated that individuals of the unsteady performer group, if their performance would have been steady (for example by more strict control or guidance), would gain as much as individuals of the steady performer group? In the end of the abstract, spatial working memory should be visuo-spatial working memory. Given the age range of the kids and the low number of children over 14 (n=2?) in this study it seems not fully justified to speak about adolescents if it comes to the effects of working memory training.", "responses": [ { "c_id": "1082", "date": "14 Nov 2014", "name": "Fiona Pugin", "role": "Author Response", "response": "Dear Professor van der Zee,We would like to thank you for your helpful comments on the manuscript. We have addressed all of them in our point by point reply below.It is not made clear enough what the passive control group is doing (or was instructed NOT to do) and particularly how this was controlled for.The subjects of the control group did not receive the means to train (laptop computer with training software installed). They were instructed to follow their habitual daily routines during the 3 weeks (during which the training group subjects conducted their training).We added this information in the ‘Materials and methods / Procedure‘ section:“The control group (N = 15) did not receive any means to perform the training and were instructed to follow their habitual activities during the three weeks. They did not have access to the training task. However, they were offered to conduct the visuospatial training after the study had been finished.” Although Table 1 is of help, the memory training compliance was said to be evaluated by a research assistant. Can more information on this evaluation be provided to convince the reader that it is likely that the compliance of the children was indeed as planned?The training software (‘Brain Twister’) stores the training record of the individual subjects. Figure 2 displays the training compliance of the subjects by indicating the number of training sessions which were conducted by the individuals during the training period of three weeks (x-axis). We extended the relevant sentence as follows:“During this visit, the working memory training compliance was evaluated by checking the training record stored on the training computer (see Figure 2 for an overview of the number of training session performed by individual subjects).” Can more be said about the cause of the individual differences in training performance (the steady and unsteady performer groups)? Is it anticipated that individuals of the unsteady performer group, if their performance would have been steady (for example by more strict control or guidance), would gain as much as individuals of the steady performer group?This is an interesting question; however, we have no data to inquire more about the cause of performance differences. To answer the question if a stricter and continuous control of the training compliance would have an influence on the steadiness of the performance, further studies are required. We added the following to the ‘Discussion‘ section:“However, the influencing factors for a steady performance increase are not known. Further studies may show if such differences in training performance may be diminished by enhancing the individual’s motivation or by an improved guidance and control through the daily training sessions.” In the end of the abstract, spatial working memory should be visuo-spatial working memory.This issue was corrected. Given the age range of the kids and the low number of children over 14 (n=2?) in this study it seems not fully justified to speak about adolescents if it comes to the effects of working memory training.We appreciate this comment (there are two subjects over 14 years in the training group and four subjects in the control group). Accordingly, we changed the wording in the title as well as in the text by limiting our statements such that they refer to children only.New Title: “Working memory training shows immediate and long-term effects on cognitive performance in children”Changes in the abstract:“Our aim was to examine not only immediate but also long-term effects of intensive working memory training on cognitive performance tests in children.”“We conclude that visuospatial working memory training in children boosts performance in similar memory tasks such as the auditory n-back task.”Change in the discussion:“We conclude from these results that the performance increase in a visuospatial working memory task is beneficial for auditory working memory in children”." } ] } ]	1	https://f1000research.com/articles/3-82
https://f1000research.com/articles/3-320/v1	31 Dec 14	{ "type": "Method Article", "title": "Parallel DNA polymerase chain reaction: Synthesis of two different PCR products from a DNA template", "authors": [ "Vikash Bhardwaj", "Kulbhushan Sharma", "Kulbhushan Sharma" ], "abstract": "Conventionally, in a polymerase chain reaction (PCR), oligonucleotide primers bind to the template DNA in an antiparallel complementary way and the template DNA is amplified as it is. Here we describe an approach in which the first primer binds in a parallel complementary orientation to the single-stranded DNA, leading to synthesis in a parallel direction. Further reactions happened in a conventional way leading to the synthesis of PCR product having polarity opposite to the template used. This is the first study showing that synthesis of DNA can happen also in a parallel direction. We report that from a single-stranded DNA template, two different but related PCR products can be synthesized.", "keywords": [ "Our fundamental knowledge of DNA structure is based on the Watson-Crick model of DNA double helix", "in which two polynucleotide chains running in opposite direction are held together by hydrogen bonds between the nitrogenous bases. Guanine can bind specifically only to cytosine (G-C) whereas adenine can bind specifically to thymine (A-T). These reactions are described as base pairing and the paired bases are said to be “complementary”1. Conformational polymorphism of DNA is now extending beyond the Watson-Crick double helix. In 1986", "using forced field calculation for a short ‘A-T’ rich DNA", "Pattabiraman proposed the hypothesis that homopolymeric duplex DNA containing d(A)6d.(T)6 can form a thermodynamically stable parallel right-handed duplex DNA with reverse Watson-Crick base pairing. He also reported that the number and type of hydrogen bonds between A-T base pair are the same as that of antiparallel double helix2. In 1988", "the experimental strategies by Ramsing and Jovin confirmed that DNA containing A-T base pairs can exist as a stable parallel-stranded helix. The “Tm” value of both PS-DNA (parallel-stranded DNA) and APS-DNA (antiparallel-stranded DNA) showed a classical dependence upon salt concentration. They reported that at any given NaCl concentration", "the melting temperature of PS-DNA was 15°C lower than its APS-DNA counterpart. In 2 mM MgCl2", "the melting temperature for PS-DNA and APS-DNA was reported approximately same as those obtained in 0.2–0.3 M NaCl", "demonstrating pronounced stabilization afforded by divalent cations3. A similar study by Sande et al. on hairpin deoxyoligonucleotides having oligonucleotides sequence in parallel polarities (PS-hairpin) also confirmed the existence of parallel-stranded conformation. They have shown that parallel-stranded hairpins form stable duplex and get denatured at 10°C lower than corresponding APS oligomers4. These two experimental studies provided evidence that DNA containing “A-T” base pairs can form both PS-DNA and APS-DNA. In 1992", "Tchurikov et al. showed that parallel complementary probes of normal nucleotide consisting of both AT/GC base pairs can be used for molecular hybridization experiments", "indicating the stability of G-C containing parallel DNA5. In 1993", "Borisova et al. reported that G-C pairs in a 40 base pair parallel duplex DNA (consisting of natural DNA sequence) are more thermostable than A-T base pairs6. Furthermore", "other similar reports have shown that there are no drastic differences in nearest neighbor base pair interactions between PS-DNA and APS-DNA having mixed AT/GC composition7. The specificity of the interaction between the strands in parallel DNA has also been studied and it is so high that parallel probe as short as 40 nucleotide length is able to detect a specific band in Southern blot hybridizations on whole genome DNA8. The polymerase chain reaction (PCR) developed by Mullis consists of denaturation of double-stranded DNA", "primer annealing and extension. The process is repeated multiple times and the template DNA is amplified millions of times without any change in polarity of DNA9 (Figure 1). In 2000", "Veitia and Ottolenghi reported that several attempts to amplify L15253 by PCR using different pairs of primers were unsuccessful. They suggested that there are no thermodynamic constraints which will prevent parallel nucleic acid synthesis", "and the deoxynucleotide triphosphates used for a normal antiparallel polymerization reaction can also serve for a parallel reaction", "provided that the polymerase enzyme is capable in catalyzing the nucleophilic interaction between the 3´OH and a 5´PPP from nucleotides arranged in a parallel way with respect to the template DNA10." ], "content": "Introduction\n\nOur fundamental knowledge of DNA structure is based on the Watson-Crick model of DNA double helix, in which two polynucleotide chains running in opposite direction are held together by hydrogen bonds between the nitrogenous bases. Guanine can bind specifically only to cytosine (G-C) whereas adenine can bind specifically to thymine (A-T). These reactions are described as base pairing and the paired bases are said to be “complementary”1. Conformational polymorphism of DNA is now extending beyond the Watson-Crick double helix. In 1986, using forced field calculation for a short ‘A-T’ rich DNA, Pattabiraman proposed the hypothesis that homopolymeric duplex DNA containing d(A)6d.(T)6 can form a thermodynamically stable parallel right-handed duplex DNA with reverse Watson-Crick base pairing. He also reported that the number and type of hydrogen bonds between A-T base pair are the same as that of antiparallel double helix2. In 1988, the experimental strategies by Ramsing and Jovin confirmed that DNA containing A-T base pairs can exist as a stable parallel-stranded helix. The “Tm” value of both PS-DNA (parallel-stranded DNA) and APS-DNA (antiparallel-stranded DNA) showed a classical dependence upon salt concentration. They reported that at any given NaCl concentration, the melting temperature of PS-DNA was 15°C lower than its APS-DNA counterpart. In 2 mM MgCl2, the melting temperature for PS-DNA and APS-DNA was reported approximately same as those obtained in 0.2–0.3 M NaCl, demonstrating pronounced stabilization afforded by divalent cations3. A similar study by Sande et al. on hairpin deoxyoligonucleotides having oligonucleotides sequence in parallel polarities (PS-hairpin) also confirmed the existence of parallel-stranded conformation. They have shown that parallel-stranded hairpins form stable duplex and get denatured at 10°C lower than corresponding APS oligomers4. These two experimental studies provided evidence that DNA containing “A-T” base pairs can form both PS-DNA and APS-DNA. In 1992, Tchurikov et al. showed that parallel complementary probes of normal nucleotide consisting of both AT/GC base pairs can be used for molecular hybridization experiments, indicating the stability of G-C containing parallel DNA5. In 1993, Borisova et al. reported that G-C pairs in a 40 base pair parallel duplex DNA (consisting of natural DNA sequence) are more thermostable than A-T base pairs6. Furthermore, other similar reports have shown that there are no drastic differences in nearest neighbor base pair interactions between PS-DNA and APS-DNA having mixed AT/GC composition7. The specificity of the interaction between the strands in parallel DNA has also been studied and it is so high that parallel probe as short as 40 nucleotide length is able to detect a specific band in Southern blot hybridizations on whole genome DNA8. The polymerase chain reaction (PCR) developed by Mullis consists of denaturation of double-stranded DNA, primer annealing and extension. The process is repeated multiple times and the template DNA is amplified millions of times without any change in polarity of DNA9 (Figure 1). In 2000, Veitia and Ottolenghi reported that several attempts to amplify L15253 by PCR using different pairs of primers were unsuccessful. They suggested that there are no thermodynamic constraints which will prevent parallel nucleic acid synthesis, and the deoxynucleotide triphosphates used for a normal antiparallel polymerization reaction can also serve for a parallel reaction, provided that the polymerase enzyme is capable in catalyzing the nucleophilic interaction between the 3´OH and a 5´PPP from nucleotides arranged in a parallel way with respect to the template DNA10.\n\nIn this study, we explored whether parallel DNA synthesis is feasible. We proposed the hypothesis that this reaction can be possible if we start a reaction using single stranded DNA as a template. We have shown that the Taq DNA polymerase can even extend the oligonucleotide primer annealed to single stranded DNA in a parallel complementary manner. The details of how our proposed parallel DNA PCR differs from the conventional PCR is shown in Figure 1 and Figure 2.\n\nThe second primer binds to the newly synthesized DNA in an antiparallel manner and later both primers amplify the new DNA in a conventional manner. PCR products obtained will have opposite polarity as compared to the template used.\n\n\nMaterials and methods\n\nPAGE purified single stranded DNA of 120 bp was commercially obtained at a scale of 1 O.D. from Sigma Aldrich, USA. PCR oligonucleotide primers were also purchased at a scale of 0.05 O.D. from Sigma Aldrich. The sequence of custom synthesized template DNA and oligonucleotide primers used in the study are shown in Table 1. In the PD-PCR reaction, we used (PD-PCR-1) and (PD-PCR-2) primer set while for conventional PCR we used (PCR-1) and (PCR-2) primers (see Table 1). Rest of the reaction remained same. The details of PCR reaction mix were as follows: total reaction mix=50µl, primers=1µl each (50 picomole), Taq DNA polymerase=0.5µl (5U/µl), dNTP mix=0.5µl (10mM), 10X PCR buffer=5µl, water=39µl and template DNA=3µl (0.114 ng). Taq DNA polymerase (M0273S) and dNTP mix (N0447S) were purchased from NEB (New England Biolabs). PCR analysis was performed using Veriti® Thermal Cycler (Applied Biosystem) by taking single stranded template DNA and amplifying it for 30 cycles at varying annealing temperature viz. 45°C, 50°C, 55°C, 58°C, 60°C, 65°C. PCR programming included 30 cycles of denaturation at 95°C for 15 seconds, annealing at varying temperatures for 30 seconds (as explained above) and extension at 72°C for 30 seconds.\n\nPCR products obtained in two reactions were separated on 1% agarose gel containing ethidium bromide and were run and observed in gel-doc (DNR Bioimaging system, Jerusalem, Israel) under UV. Electrophoresis apparatus used in these experiments was purchased from Chromus Biotech, Bengaluru, India whereas chemicals {Agarose (A9539) and EtBr (E7637)} were purchased from Sigma, USA.\n\nThe amplified products were sequenced at Eurofins Genomics India Pvt. Ltd. Karnataka India.\n\nReal time PCR was performed with 2X SYBR Green master mix (K0221, Thermo Scientific, Pittsburgh, USA). The details of real time PCR reaction mix were as follows: total reaction mix=10µl, primers=0.25µl each, (50 picomole), 2X SYBR green mastermix=5µl, water=4µl and template=0.5µl (1:1000 dilution from original stock of 0.96 picomole). In the PD-PCR reaction, we used (PD-PCR-1) and (PD-PCR-2) primer set while for conventional PCR we used (PCR-1) and (PCR-2) primers (see Table 1). Negative control included reaction mix without template DNA. Reactions were incubated at 94°C for 5 minutes, followed by 30 PCR cycles of 94°C for 15 seconds, 50°C for 30 seconds and 72°C for 60 seconds using Mx3005P qPCR System - Agilent Technologies, Inc. The data were analyzed by using 2−ΔCt method. The products were also run on agarose gel and visualised on gel doc system as previously described.\n\n\nResult and discussion\n\nThe thermal denaturation analysis of parallel DNA has shown that it melts at a lower temperature than the corresponding antiparallel structure3,4. This finding gives us the clue that using double-stranded antiparallel DNA as a template for PD-PCR will not be possible as during annealing steps, antiparallel double-stranded DNA will anneal to itself without binding to parallel-stranded complementary primers. To avoid this, we started our PCR with a single-stranded DNA template. Details on how our proposed parallel DNA PCR (PD-PCR) differs from conventional PCR are shown in Figure 2. The first oligonucleotide primer (PD-PCR-1) was designed to bind the single-stranded template DNA in a parallel complementary manner. The parallel complementary annealing of the first primer allowed the synthesis of DNA in a parallel direction to the single-stranded DNA template. After the first denaturation step, the second oligonucleotide primer (PD-PCR-2) was designed to anneal to the newly synthesized DNA in an antiparallel complementary orientation. Further, both first and second primers used in this reaction amplified the new second DNA strand in a conventional way by binding in an antiparallel complementary way. Figure 3 (lanes 8–13) shows a 120 bp PCR product amplified by parallel DNA PCR scheme at annealing temperature of 45°C, 50°C, 55°C, 58°C, 60°C, 65°C respectively. In all cases, denaturation was performed at 95°C for 15 seconds, annealing for 30 seconds while extension at 72°C for 30 second for a total of 30 cycles. Similarly, as a control reaction, the single-stranded 120 bp DNA was amplified by conventional PCR in which the first primer (PCR-1) bound to the template DNA in an antiparallel orientation and the second primer (PCR-2) annealed to the newly synthesized DNA in an antiparallel orientation. Figure 3, Lanes 1–6 shows a 120 bp product PCR amplified at annealing temperature of 45°C, 50°C, 55°C, 58°C, 60°C, 65°C respectively using conventional antiparallel complementary primers. As a control reaction, PD-PCR was also performed using only one of the two primers. As expected, no PCR products were obtained (Figure 4A lane 2 and 3). As a control reaction, conventional PCR and PD-PCR were performed without adding any template DNA. As expected, no PCR product was obtained confirming that no primer dimer was formed during both conventional PCR and PD-PCR (Figure 4B). The DNA sequencing results confirmed that DNA templates were amplified in two different PCR products. Conventional PCR amplified the template DNA in its original orientation (Figure 5A) whereas PD-PCR products read in a parallel direction to the template DNA (Figure 5B). Primers and template used to show feasibility of PD-PCR till now (Figure 3 and Table 1) were further used to perform real-time PCR. For this, a master mix containing SYBR Green and other components (except template and primers) was used. Primers and template were added to the master mix to make a final volume of 10µl. A Ct value of 9.26 was obtained for conventional PCR, 23.29 for PD-PCR whereas 33.15 was observed in negative control (without adding template DNA) indicating amplification in both conventional PCR and PD-PCR reactions (Figure 6). The amplification indicated by Ct value in real time PCR was also confirmed by running the product on agarose gel (Figure 6, lower panel). Weak amplifications in PD-PCR may be attributed to the fact that the actual amplification in conventional PCR is one step ahead than the PD-PCR. The first amplification in conventional PCR starts as early as denaturation followed by annealing (Figure 1). On the other hand, in PD-PCR a new template is first synthesized during the first amplification (represented by green color in Figure 2). Once the template is ready, the conventional PCR goes on. Therefore, the amplified product shows low intensity as compared to the conventional PCR products. Taking together, our study has shown that DNA synthesis can happen in a parallel direction and two different, but related PCR products can be synthesized from the single-stranded template DNA. We hope that more molecular biology techniques will develop in future based on parallel complementary bindings of duplex DNA.\n\nLane 7 is 100 bp molecular weight marker and Lanes 8–13 show PCR products amplified by parallel DNA PCR scheme at annealing temperature of 45°C, 50°C, 55°C, 58°C, 60°C, 65°C, respectively. In all cases, denaturation was performed at 95°C for 15 seconds, annealing for 30 seconds while extension at 72°C for 30 second for a total of 30 cycles.\n\n(A): A control reaction showing that PCR products were obtained when both primers were added as per scheme in Figure 2. In Figure 4 (A), Lane 1 shows 120 bp PCR products synthesized by PD-PCR, while in Lanes 2 and 3, only single primers were added and as expected no PCR product was synthesized. Figure 4(B) shows a negative control reaction of conventional PCR and PD-PCR in which the template DNA was not added.\n\nDNA sequencing results. Sequencing results in (A) show that 120 bp DNA was amplified as it is while sequencing results in (B) confirm that PCR products were obtained as per the scheme shown in Figure 2.\n\nReal time PCR and PD-PCR (A) show ampliflication plot and dissociation curves obtained after real time PCR analysis of amplification of 120 nucleotides single stranded template DNA via conventional PCR and PD-PCR. In control reaction no template DNA was added. (B) PCR products obtained in real time PCR were also run on agarose gel and visualised on gel doc system.\n\n\nData availability\n\nF1000Research: Dataset 1. DNA sequencing file for PCR and PD-PCR. 10.5256/f1000research.5813.d4151511\n\nF1000Research: Dataset 2. Real time PCR file for PCR and PD-PCR. 10.5256/f1000research.5813.d4151612", "appendix": "Author contributions\n\n\n\nVB and KS designed the experiment. VB and KS carried out the research. Both prepared the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nWe are thankful to Harpreet Singh (Territory Manager, Sigma-Aldrich, Gujarat, India) for providing the custom synthesized template DNA and oligonucleotide primers used in this study.\n\n\nReferences\n\nWatson JD, Crick FH: Molecular structure of nucleic acids; a structure for Deoxyribose Nucleic Acid. Nature. 1953; 171(4356): 737–738. PubMed Abstract \| Publisher Full Text\n\nPattabiraman N: Can the Double Helix Be Parallel? Biopolymers. 1986; 25(9): 1603–1606. PubMed Abstract \| Publisher Full Text\n\nRamsing NB, Jovin TM: Parallel stranded duplex DNA. Nucleic Acids Res. 1988; 16(14A): 6659–76. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan de Sande JH, Ramsing NB, Germann MW, et al.: Parallel Stranded DNA. Science. 1988; 241(4865): 551–557. PubMed Abstract \| Publisher Full Text\n\nTchurikov NA, Shchyolkina AK, Borissova OF, et al.: Southern molecular hybridization experiments with parallel complementary DNA probes. FEBS Lett. 1992; 297(3): 233–236. PubMed Abstract \| Publisher Full Text\n\nBorisova OF, Shchyolkina AK, Chernov BK, et al.: Relative stability of AT and GC pairs in parallel DNA duplex formed by a natural sequence. FEBS Lett. 1993; 322(3): 304–6. PubMed Abstract \| Publisher Full Text\n\nShchyolkina AK, Borisova OF, Livshits MA, et al.: [Parallel-stranded DNA with natural base sequences]. Mol Biol. 2003; 37(2): 223–231. PubMed Abstract \| Publisher Full Text\n\nTchurikov NA, Shchyolkina AK, Borissova OF, et al.: Southern molecular hybridization experiments with parallel complementary DNA probes. FEBS Lett. 1992; 10: 297(3): 233–6. PubMed Abstract \| Publisher Full Text\n\nMullis KB: Process for amplifying nucleic acid sequences, United States Patent 4683202. 1987. Reference Source\n\nVeitia R, Ottolenghi C: Placing parallel stranded DNA in an evolutionary context. J Theor Biol. 2000; 206(2): 317–322. PubMed Abstract \| Publisher Full Text\n\nBhardwaj V, Sharma K: Dataset 1. DNA sequencing file for PCR and PD-PCR. F1000Research. 2014. Data Source\n\nBhardwaj V, Sharma K: Dataset 2. Real time PCR file for PCR and PD-PCR. F1000Research. 2014. Data Source" }	[ { "id": "7172", "date": "12 Jan 2015", "name": "Harishkumar Madhyastha", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title of the research paper is well justified and suitable to the content of the subject. Abstract of the paper is well written and concluded. Materials and method, data analysis and design of the experiment are commendable. In my opinion, the paper is well written and opens a new paradigm for researchers and finally, the conclusion arrived at shows the high standard of the scientific achievement. In all respects, the paper is highly novel and can be indexed.", "responses": [] }, { "id": "7174", "date": "19 Jan 2015", "name": "Ram Gopal", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is interesting to read the article ‘Parallel DNA polymerse chain reaction: Synthesis of two different PCR products from a DNA template’ by Bhardwaj and Sharma. The authors for the first time have demonstrated that primers can anneal in parallel orientation and can be extended in a PCR reaction. They have used state of the art technique real time PCR to demonstrate this along with the conventional agarose gel electrophoresis and DNA sequencing reaction. Definitely, this opens up many possibilities in the field of molecular biology. The research is well designed and presented in a clear and comprehensible way. The introduction section gives a very nice insight. However, I suggest following minor changes to improve the manuscript. Figure 3- Numbering should be done on the loading wells. Figure 5- Sequencing results should be presented in such a way that one can easily compare the APS and PS sequences easily. For example they can put a arrow mark from where to compare. Figure 6- It is difficult to read the abscissa and ordinate scale and naming. Increasing the font will help. Line 11- d(A)6d.(T)6 should be explained or is it dA6 dT6 ? In the RT PCR section the term Ct value should be explained so that a non-expert reader can benefit. There are some typos errors scattered in the manuscript. Such as 50µl should be 50 µL.", "responses": [] } ]	1	https://f1000research.com/articles/3-320
https://f1000research.com/articles/3-318/v1	30 Dec 14	{ "type": "Case Report", "title": "Case Report: Bilateral reexpansion pulmonary edema following treatment of a unilateral hemothorax", "authors": [ "Steven P de Wolf", "Jaap Deunk", "Alexander D Cornet", "Paul WG Elbers", "Steven P de Wolf", "Jaap Deunk", "Alexander D Cornet" ], "abstract": "Bilateral re-expansion pulmonary edema (RPE) is an extremely rare entity. We report the unique case of bilateral RPE following a traumatic, unilateral hemopneumothorax in a young healthy male. Bilateral RPE occurred only one hour after drainage of a unilateral hemopneumothorax. The patient was treated with diuretics and supplemental oxygen. Diagnosis was confirmed by excluding other causes, using laboratory findings, chest radiography, pulmonary and cardiac ultrasound and high resolution computed tomography. His recovery was uneventful. The pathophysiology of bilateral RPE is not well known. Treatment is mainly supportive and consists of diuretics, mechanical ventilation, inotropes and steroids. In case of a pulmonary deterioration after the drainage of a traumatic pneumothorax, bilateral RPE should be considered after exclusion of more common causes of dyspnea.", "keywords": [ "Re-expansion pulmonary edema", "bilateral", "hemothorax", "pneumothorax", "trauma" ], "content": "Introduction\n\nWe report here on a unique case of bilateral re-expansion pulmonary edema (RPE). First described in 1958, RPE is a rare, but well known complication of thoracocentesis1. RPE usually occurs unilaterally after expansion of the ipsilateral collapsed lung caused by either spontaneous pneumothorax or various types of pleural effusion2. However, in this case, RPE occurred bilaterally, following expansion of a unilateral hemopneumothorax in the setting of trauma.\n\n\nCase\n\nA 31-year old caucasian male with no significant past medical history was brought to our emergency department after falling 1.5 meters down from a platform. He was fully conscious and both respiratory and hemodynamically stable. Secondary survey findings included a fractured left olecranon and fractures of costae 7 to 9 on the left side, without clinical or radiological signs of a pneumothorax.\n\nAfter two days in the hospital, he underwent tension band wiring of his olecranon under general anaesthesia. There were no difficulties during mechanical ventilation. However, on the first postoperative day, his peripheral oxygen saturation was noted to be 93% without supplemental oxygen. Auscultation yielded decreased breath sounds on the left side and a chest radiograph showed a fully collapsed left lung with pleural effusion (Figure 1). A chest tube was placed which immediately drained air and 250 mL of blood.\n\nTo our surprise, follow-up chest radiography one hour after drainage, demonstrated diffuse bilateral airspace opacification, peribronchial cuffing and Kerley-B lines, indicating bilateral pulmonary edema (Figure 2). The chest tube was in a good position. In the course of several hours our patient became increasingly dyspnoeic, requiring 15 liters of oxygen via a non-rebreathing mask. He was transferred to the intensive care unit.\n\nIntensive care ultrasound showed bilateral B-lines in all lung fields (Figure 3), normal left and right ventricular function, no valvular dysfunction, normal atrial and caval vein dimensions and no pericardial effusion. These findings are consistent with non-cardiogenic pulmonary edema. Our patient did not receive excessive fluid therapy or blood transfusions and N-terminal-pro-B-type natriuretic peptide was normal (430ng/L), as were white cell count (9,6×109s/L) and C-reactive protein (23mg/L). Through this process of exclusion, and consistent with recent lung re-expansion, our patient was diagnosed with bilateral RPE.\n\nAggressive diuretic therapy markedly improved his dyspnea without the need for mechanical ventilation and our patient was transferred back to the ward after 24 hours. Because of a persisting dependency of supplemental oxygen, high resolution computed tomography was performed two days later. This confirmed our diagnosis of bilateral pulmonary edema and revealed two additional rib fractures on the left side. Diuretics and oxygen suppletion were discontinued after a few days, and twelve days after the initial trauma our patient was discharged to home.\n\nThe dotted arrows indicate the rib shadows. The horizontal arrow indicates the pleura. Between the dotted arrows B-lines can be seen in a pattern called ground-glass rockets, showing an interstitial syndrome.\n\n\nDiscussion\n\nTo the best of our knowledge, this is the first report of bilateral RPE following thoracocentesis of a unilateral traumatic hemopneumothorax. A few cases of bilateral RPE have been described in literature3–11. However, none of these cases were preceded by a traumatic injury. In fact, most reported cases of either unilateral or bilateral RPE followed non-traumatic pneumothorax, pleural empyema or pleural effusion. The incidence of unilateral RPE is between 0 and 6,5% whereas bilateral RPE is extremely rare11–14.\n\nThe pathophysiology of bilateral RPE is not well known. Increased levels of the pro-inflammatory cytokine interleukin-8 and monocyte chemo-attractant protein 1 might be involved in the inflammatory process that characterizes RPE15. In addition, re-expansion of the lung may lead to reperfusion injury and increased permeability of the endovascular cells16. A prolonged collapse seems to result in an increased risk for RPE4,11. Other risk factors include the extent of lung collapse, young age17 and fast re-expansion using suction4. Treatment is still mainly supportive and relies mostly on diuretics but may necessitate mechanical ventilation, inotropes and steroids11,17.\n\nIn conclusion, bilateral re-expansion pulmonary edema is an extremely rare but fascinating phenomenon following treatment of a unilateral traumatic hemopneumothorax. In case of a pulmonary deterioration after the drainage of a traumatic pneumothorax, bilateral RPE should be considered, after exclusion of more common causes of dyspnea.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the patient.", "appendix": "Author contributions\n\n\n\nSdW, JD and PE contributed to data acquisition. SdW prepared the first draft of the manuscript. JD, AC and PE contributed to manuscript drafting.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in the funding of this work.\n\n\nReferences\n\nCarlson RI, Classen KL, Gollan F, et al.: Pulmonary edema following the rapid reexpansion of a totally collapsed lung due to a pneumothorax: a clinical and experimental study. Surg Forum. 1958; 9: 367–371. PubMed Abstract\n\nNeustein SM: Reexpansion pulmonary edema. J Cardiothorac Vasc Anesth. 2007; 21(6): 887–891. PubMed Abstract \| Publisher Full Text\n\nHenderson AF, Banham SW, Moran F: Re-expansion pulmonary oedema: a potentially serious complication of delayed diagnosis of pneumothorax. Br Med J (Clin Res Ed). 1985; 291(6495): 593–594. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMahfood S, Hix WR, Aaron BL, et al.: Reexpansion pulmonary edema. Ann Thorac Surg. 1988; 45(3): 340–345. PubMed Abstract \| Publisher Full Text\n\nRagozzino MW, Green R: Bilateral reexpansion pulmonary edema following unilateral pleurocentesis. Chest. 1991; 99(2): 506–508. PubMed Abstract \| Publisher Full Text\n\nÖzlü O, Kilic A, Cengizlier R: Bilateral re-expansion pulmonary edema in a child: a reminder. Acta Anaesthesiol Scand. 2000; 44(7): 884–885. PubMed Abstract \| Publisher Full Text\n\nDubose J, Perciballi J, Timmer S, et al.: Bilateral reexpansion pulmonary edema after treatment of spontaneous pneumothorax. Curr Surg. 2004; 61(4): 376–379. PubMed Abstract\n\nBaik JH, Ahn MI, Park YH, et al.: High-resolution CT findings of re-expansion pulmonary edema. Korean J Radiol. 2010; 11(2): 164–168. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaksu MS, Paksu S, Akgün M, et al.: Bilateral reexpansion pulmonary edema associated with pleural empyema: a case report. Eur J Pediatr. 2011; 170(9): 1205–1207. PubMed Abstract \| Publisher Full Text\n\nGleeson T, Thiessen R, Müller N: Reexpansion Pulmonary edema computed tomography findings in 22 patients. J Thorac Imaging. 2011; 26(1): 36–41. PubMed Abstract \| Publisher Full Text\n\nHaga T, Kurihara M, Kataoka H: The risk for re-expansion pulmonary edema following spontaneous pneumothorax. Surg Today. 2013; 44(10): 1823–7. PubMed Abstract \| Publisher Full Text\n\nMills M, Balsch BF: Spontaneous pneumothorax: A series of 400 cases. Ann Thorac Surg. 1965; 122: 286–297. PubMed Abstract \| Publisher Full Text\n\nBrooks JW: Open thoracotomy in the management of spontaneous pneumothorax. Ann Surg. 1973; 177(6): 798–805. PubMed Abstract \| Free Full Text\n\nRozenman J, Yellin A, Simansky DA, et al.: Re-expansion pulmonary oedema following spontaneous pneumothorax. Respir Med. 1996; 90(4): 235–238. PubMed Abstract \| Publisher Full Text\n\nSakao Y, Kajikawa O, Martin TR, et al.: Association of IL-8 and MCP-1 with the development of reexpansion pulmonary edema in rabbits. Ann Thorac Surg. 2001; 71(6): 1825–1832. PubMed Abstract \| Publisher Full Text\n\nSivrikoz MC, Tunçözgür B, Cekmen M, et al.: The role of tissue reperfusion in the reexpansion injury of the lungs. Eur J Cardiothorac Surg. 2002; 22(5): 721–727. PubMed Abstract \| Publisher Full Text\n\nMatsuura Y, Nomimura T, Murakami H, et al.: Clinical analysis of reexpansion pulmonary edema. Chest. 1991; 100(6): 1562–1566. PubMed Abstract \| Publisher Full Text" }	[ { "id": "7215", "date": "06 Jan 2015", "name": "André Coetzee", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI approve of this submission. It is a rare event and the authors rather succinctly highlight the uncertainties and possible mechanisms. The post X ray is rather stunning and without the history this could indeed be confusing!", "responses": [] }, { "id": "7695", "date": "18 Feb 2015", "name": "Ehab Farag", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI approve this case report. It is well written and informative one.", "responses": [] } ]	1	https://f1000research.com/articles/3-318
https://f1000research.com/articles/3-314/v1	23 Dec 14	{ "type": "Case Report", "title": "Case Report: Large nested variant urothelial carcinoma –invasive malignancy masquerading as low grade disease", "authors": [ "Andrew Keller", "Ai Jye Lim", "Ahmad Ali", "Ai Jye Lim", "Ahmad Ali" ], "abstract": "IntroductionThe large nested variant of urothelial carcinoma (LNVUC) is a newly described and rare subtype of urothelial carcinoma. It is characterised by bland cytological features and a large nested architecture similar in appearance to low grade urothelial carcinoma with an inverted growth pattern. To date only 23 cases in a single series have been described. Case ReportWe describe the case of a 59 year old male with LNVUC whose tumour was initially misdiagnosed as a non-invasive low grade urothelial carcinoma. At a subsequent re-resection, his tumour was correctly re-classified as LNVUC with extensive invasion of the muscularis propria. Radical cystectomy and formation of an ileal conduit was performed. His operative specimen revealed invasion of prostatic stroma and perivesical fat, with all surgical margins clear. He is currently free from clinical recurrence 12 months after his cystectomy. ConclusionLNVUC is a newly described and rare urothelial carcinoma subtype. It characteristically possesses bland cytological features and may mimic low grade urothelial cancer. Despite its bland appearance it behaves aggressively with invasion, metastasis and death being common.", "keywords": [ "LNVUC", "Urothelial carcinoma" ], "content": "Introduction\n\nThe large nested variant of urothelial carcinoma (LNVUC) is a newly described variant of urothelial cancer (UC), with a single series of 23 cases being the only examples reported thus far1. This aggressive UC variant has deceptively bland cytological features, which may confound correct tumour classification. We present the case of a 59 year old male with a large bladder tumour who was initially diagnosed histologically as non-invasive low grade UC on initial resection. At re-resection the tumour was correctly identified as LNVUC.\n\n\nCase report\n\nA 59 year old Caucasian male was transferred to our unit from a regional hospital with a two week history of macroscopic haematuria. He sought medical attention only after he developed clot retention. He denied any previous history of haematuria or urinary problems prior to the two week period immediately before his hospital admission.\n\nHis medical history was unremarkable other than extensive carcinogen exposure, with both a 40 pack-year smoking history and significant occupational exposure, working as a fly-in, fly-out diesel fitter on a mine site.\n\nOn admission he required placement of an indwelling urinary catheter and continuous bladder irrigations. His initial serum creatinine was elevated, but soon normalised following catherisation. He was transferred to our secondary referral centre following failure of conservative therapies to control his persistent haematuria.\n\nOn his arrival to our facility we arranged Computerised Tomography (CT) to assess his bladder and upper renal tracts. CT demonstrated a grossly thick walled bladder with a large enhancing intra-vesical mass, and bilateral hydroureteronephrosis (Figure 1). His haematuria continued and he became transfusion dependant. He was taken to the operating theatre two days after his arrival for cystoscopic assessment.\n\nAt cystoscopy, there was a large papillary tumour involving the prostatic urethra, the trigone, and both lateral walls of the bladder. (Figure 2) Neither ureteric orifice was identifiable. The tumour was macroscopically resected after an extensive procedure.\n\nHistologically the tumour was classified as a low grade urothelial carcinoma with no evidence of superficial or muscle invasion. We found this finding inconsistent with the operative and radiologic findings and repeated a cystoscopy four weeks later.\n\nAt repeat cystoscopy large volume tumour regrowth had occurred and a further 90 minute resection was performed. Tumour histology this time demonstrated invasion into the muscularis propia by a large nested variant of UC (Figure 3) with an adjacent superficial component of low grade papillary UC (Figure 4).\n\nA staging Positron Emission Tomography (PET) CT was negative for metastatic disease and a cysto-prostatectomy and formation of an ileal conduit was performed. The operative specimen histology again revealed the large nested variant of UC with focal invasion into peri-vesical fat (Figure 5) and the prostatic stroma (Figure 6). A component of low grade UC was also present superfically. The tumour was clear of all operative margins. All lymph nodes sampled were negative for metastatic deposits.\n\nThe patient’s post-operative period was unremarkable and he made a swift recovery. He was discharged from hospital one week post-operatively. He was referred to medical oncology for consideration of adjuvant chemotherapy, however after discussion with oncologists the patient declined any additional treatment. He is presently twelve months post cysto-prostatectomy and he remains clinically well and free from clinical disease recurrence. We will continue to closely monitor this patient.\n\n\nDiscussion\n\nThe large nested variant is a newly described subtype of UC. The first and to date only case series was published in 2011 by Cox and Epstein and describes 23 cases1. They describe tumours with universally bland histologic appearances but with invasion of large nests resembling von Brunns nests into the underlying stroma. In contrast to the normal nested variant of UC, a surface papillary component is present and there is abundant fibrous stroma between individual tumour nests1,2. LNVUC is most commonly mistaken for low grade urothelial cancer with an inverted growth pattern2.\n\nLNVUC behaves aggressively, of the 17 cases with adequate follow-up in Cox and Epstein’s series, 3 had died of their disease and another two were alive but had developed metastatic spread of their cancer1.\n\n\nConclusion\n\nThe large nested variant is an extremely rare, newly described variant of UC. Our case is only the 24th described in the literature, and the first case reported since the condition was first classified in 2011. LNVUC can confound accurate diagnosis by masquerading as Von Brunn’s nests or, in our case, low grade non-invasive UC. Despite the bland macroscopic and histologic appearance of LNVUC it behaves in an aggressive manner, and should be treated the same as any invasive urothelial malignancy.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patient.", "appendix": "Author contributions\n\n\n\nAK was the author of the paper. AJL provided pathological input into the case report and provided images. AA contributed to writing of the article and was involved in proofing.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nCox R, Epstein JI: Large nested variant of urothelial carcinoma: 23 cases mimicking von Brunn nests and inverted growth pattern of noninvasive papillary urothelial carcinoma. Am J Surg Pathol. 2011; 35(9): 1337–42. PubMed Abstract \| Publisher Full Text\n\nSamaratunga H, Delahunt B: Recently described and unusual variants of urothelial carcinoma of the urinary bladder. Pathology. 2012; 44(5): 407–18. PubMed Abstract" }	[ { "id": "7134", "date": "07 Jan 2015", "name": "M. Hammad Ather", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nNested variant of UC is indeed rarely reported. It is however, not certain whether the actual incidence is low or it is less frequently recognized. The estimated incidence is 0.3% (Hong et al., 2007).Authors have correctly recognized the disparity in clinical parameters (Significant exposure, 40 pack year history, industrial exposure, large bladder growth and obstructive uropathy) with the pathology indicating low grade cancer (pTa LG).Most of the recognized clinical indicators of nested variant including male gender, age above 50 years, ureteral obstruction and cystoscopy showing tumor slightly raised and erythematous or nodular were present in this case.I have few queries on which i request the authors' to commentsTumor is known to have aggressive behavior, however, growth in 4 weeks needing 90 minute resection is rather surprising. Were any cold cup deep biopsies taken at the time of initial resection in view of the significant clinical findings of aggressive UC. Were biopsies from the bladder neck, prostatic fossa or para collicular area taken at the time of initial resection.", "responses": [ { "c_id": "1164", "date": "07 Jan 2015", "name": "Andrew Keller", "role": "Author Response", "response": "Thanks for your review.The described pathology in this case report is actually \"large nested UC\" which is a distinct clinical entity to \"nested UC\" and one only recently described. I agree that this entity, like \"nested UC\" is undoubtedly more common than is documented due to under reporting.In regards to your queries.1. I agree that the amount of tumour re-resection needed after only a 4 week interval following a macroscopically complete resection is surprising, it certainly was for us. One explanation is that a surgical trainee and not a consultant urologist was performing the resection, which would account for some of the additional time required.2. No cold cup biopsies were taken at the time of the initial resection as the resection was felt to have adequately sampled the muscularis propria. Indeed, there was plentiful muscle present in the initial resection specimen histologically.3. Cold cup biopsies were not taken from the bladder neck, prostatic fossa and paracollicular area at the time of the initial biopsies. However both bladder neck and prostatic fossa proximal to the prostatic utricle were involved with tumour at initial resection and these areas were consequently extensively resected with loop diathermy.Thanks again for your comments and review.Andrew Keller" } ] }, { "id": "7138", "date": "02 Feb 2015", "name": "Levent Turkeri", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a timely report on large nested variant of urothelial carcinoma. It may pose a diagnostic confusion since histological appearance may resemble more indolent forms of the disease although it certainly has a high invasive potential and may be associated with metastatic disease. Therefore, the pathologists must be aware of this variant when they evaluate surgical specimens and urologists must discuss with their pathologists the possibility of the presence of such a variant when they receive a report indicating low grade disease yet the clinical presentation is not compatible.", "responses": [] }, { "id": "7541", "date": "02 Feb 2015", "name": "Daron Smith", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis case report does an excellent job of highlighting the deceptive nature of LNVUC and the clinical importance of early detection. This aggressive and rare subtype of urothelial cancer can very easily be mis-diagnosed as a low-grade urothelial tumour. The article identifies the key demographic risk factors seen in LNVUC (male, heavy smoker, age- approx 60yrs) and highlights the clinical factors that point towards an aggressive pathology (upper tract dilatation, rapid regrowth). Early recognition is vital in allowing prompt radical treatment.In our unit we have also had a very similar case of a bladder tumour which on resection was found to contain discohesive nests of invasive urothelial carcinoma. This patient also underwent radical cystectomy and final histology confirmed T3 disease.As LNVUC can cause a diagnostic dilemma, we would be interested to learn if any immunohistochemical studies were carried out and what these showed.", "responses": [] } ]	1	https://f1000research.com/articles/3-314
https://f1000research.com/articles/3-270/v1	07 Nov 14	{ "type": "Correspondence", "title": "APOC3 may not be a predictor of risk of ischemic vascular disease in the Chinese population", "authors": [ "Liang Tang", "Zhi-Peng Cheng", "Qing-Yun Wang", "Wei Zeng", "Hui Liu", "Ying-Ying Wu", "Bei Hu", "Yu Hu", "Liang Tang", "Zhi-Peng Cheng", "Qing-Yun Wang", "Wei Zeng", "Hui Liu", "Ying-Ying Wu", "Bei Hu" ], "abstract": "The genetic background of ischemic vascular disease is actively being explored. Several studies have shown that inhibition of APOC3 significantly reduces plasma levels of apolipoprotein C3 and triglycerides. Recently, the TG and HDL Working Group and Jørgensen et al. reported that loss-of-function mutations in APOC3 are associated with decreased triglyceride levels and a reduced risk of ischemic vascular disease in European and African individuals. We performed a replication study in 4470 Chinese participants. The coding regions of APOC3 were amplified and re-sequenced. However, only synonymous and intronic variants with no functional consequences were identified. None of the loss-of-function mutations reported in European and African individuals were observed. Therefore, APOC3 may not be an ideal predictor for risk of ischemic vascular disease in the Chinese population.", "keywords": [ "ischemic vascular disease", "risk prediction", "APOC3" ], "content": "Correspondence\n\nThe genetic basis of ischemic vascular disease such as coronary artery disease is actively being explored. Most studies have focused on susceptibility factors contributing to an increased risk1, while only few studies have identified protective variants conferring reduced risk. Recently, the TG and HDL Working Group and Jørgensen et al. reported that loss-of-function mutations in APOC3 are associated with decreased triglyceride levels and a reduced risk of ischemic vascular disease in individuals of European and African ancestry2,3. Approximately 1 in 150 individuals were heterozygous carriers of at least one of the four mutations: R19X, A43T, IVS2+1G→A, and IVS3+1G→T. Heterozygous carriers for these mutations had a significantly lower incidence of ischemic vascular disease as compared to non-carriers (hazard ratio = 0.59). Triglyceride and circulating APOC3 levels in the carriers were only 61% and 54% of those in non-carriers, respectively. These critical findings prompted us to undertake a replication study in China, where over one million people are affected by cardiovascular diseases each year (http://www.nhfpc.gov.cn/).\n\nA total of 4470 unrelated Chinese participants were enrolled, including 1488 healthy controls, 1050 patients with ischemic stroke, 628 patients with coronary artery disease, and 1304 patients with venous thrombosis, which could also be exacerbated by effects on the coagulation system resulting from elevated triglyceride levels. The 1488 healthy controls and 1050 patients with ischemic stroke were described in a previous study4. Briefly, healthy individuals did not present any relevant medical history or family history of ischemic vascular disease. Ischemic stroke was confirmed by brain computed tomography (CT) and/or brain magnetic resonance imaging (MRI). The 1304 patients with venous thrombosis were described previously5. Thrombosis was confirmed by objective investigations such as color Doppler ultrasonography and/or CT angiography. Patients with coronary artery disease were enrolled in our hospital from September 2013 to March 2014. Coronary artery disease was validated by angiographic evidence of at least one segment of a major coronary with over 50% organic stenosis. The characteristics of the 628 patients with coronary artery disease are summarized in Table 1. Written informed consent was obtained from all participants, and the study was approved by the Ethics Committee of Union Hospital affiliated with Huazhong University of Science and Technology (Approval number 2013-03-0052).\n\nTC, total cholesterol; TG, triglycerides; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol.\n\nThe diagnosis of myocardial infarction was based on typical chest pain with a duration over 30 min, on electrocardiographic patterns, and on increased creatine kinase MB isoenzyme and troponin I levels. Hypertension is defined as systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg. Type 2 diabetes were clarified using the 1999 WHO criteria, including fasting plasma glucose ≥ 7.0 mmol/L, 2-hour oral glucose tolerance test plasma glucose ≥ 11.1 mmol/L or ongoing therapy for diabetes.\n\nBlood samples were collected into a vacutainer tube containing 0.105 mol/L trisodium citrate and were then centrifuged at 2000 g for 15 minutes. Genomic DNA was isolated using a salt precipitation method and was then used for sequencing. The four exons and the flanking intronic regions of APOC3 were amplified by PCR and then sequenced on an Applied Biosystems ABI 3730 Genetic Analyzer, as previously described2. The oligonucleotide pairs and annealing temperatures employed in PCR and sequencing are shown in Table 2. In this study, we identified only synonymous and intronic variants, with no functional consequences, and similar genotype distributions across the groups (Table 3). None of the loss-of-function mutations reported in European and African individuals were observed in the current cohort. Considering the relatively large sample size, we suggest that functional variants in APOC3 could be very rare in China. Therefore, the genetic background of ischemic vascular disease is highly variable among different ethnic groups, and APOC3 may not be an ideal predictor of risk of ischemic vascular disease in the Chinese population. Further studies are warranted to understand the genetic basis governing triglyceride levels and conferring protective effects on ischemic vascular disease in the Chinese population.\n\nAT, annealing temperature. The accession number of APOC3 reference sequence in GenBank is NG_008949.1.\n\ndbSNP, single nucleotide polymorphism database of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/projects/SNP).\n\nComparisons between the controls and each case group were assessed with the use of the chi-square test. A two tailed P<0.05 was considered significant.\n\n\nConsent\n\nWritten informed consent to publish these data has been obtained by each participant.", "appendix": "Author contributions\n\n\n\nYH and LT chose the article for correspondence and evaluated the data in the manuscript. LT, ZPC, QYW, WZ, HL, YYW, and BH performed experiments. LT and ZPC wrote the manuscript. YH supervised the process and critically edited the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was supported by grants from the National Natural Sciences Foundation of China (No. 81370622 and No. 81400099) and the Independent Innovation Foundation of Huazhong University of Science and Technology (No. 01-18-530045, 2013QN213).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nPeden JF, Farrall M: Thirty-five common variants for coronary artery disease: the fruits of much collaborative labour. Hum Mol Genet. 2011; 20(R2): R198–205. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJørgensen AB, Frikke-Schmidt R, Nordestgaard BG, et al.: Loss-of-function mutations in APOC3 and risk of ischemic vascular disease. N Engl J Med. 2014; 371(1): 32–41. PubMed Abstract \| Publisher Full Text\n\nThe TG and HDL Working Group of the Exome Sequencing Project, National Heart, Lung, and Blood Institute: Loss-of-function mutations in APOC3, triglycerides, and coronary disease. N Engl J Med. 2014; 371(1): 22–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLu X, Tang L, Xu K, et al.: Novel association of a PROC variant with ischemic stroke in a Chinese Han population. Hum Genet. 2013; 132(1): 69–77. PubMed Abstract \| Publisher Full Text\n\nTang L, Wang HF, Lu X, et al.: Common genetic risk factors for venous thrombosis in the Chinese population. Am J Hum Genet. 2013; 92(2): 177–187. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "6688", "date": "13 Nov 2014", "name": "Javier Corral", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDespite being a negative study, the information in this article is valuable. It describes a study that found no relevant mutation in APOC3 among a large number of Chinese subjects (4470), including 1488 healthy controls, 1050 patients with ischemic stroke, 628 patients with coronary artery disease, and 1304 patients with venous thrombosis. Actually, no one with the loss-of-function mutations reported in European and African individuals was identified in this study. Only synonymous and intronic variants were discovered. The authors indicated that these variants have no functional consequences, but no data are shown. At the least the TG levels according to the genotype might be shown in table 2. The authors must indicate that other genetic defects, such a gross deletions or regulatory mutations not identified by sequencing methods or by analysis of exons, may cause a loss-of-function of APOC3 and might be associated with decreased triglyceride levels and a reduced risk of ischemic vascular disease. Actually, selection of subjects with decreased triglyceride levels and a deeper analysis of this gene might be a better strategy to identify these genetic variants potentially involved in TG levels and risk of ischemic vascular disease. Accordingly, I think the authors might change the title of the article.", "responses": [] }, { "id": "6920", "date": "05 Dec 2014", "name": "Chang-Geng Ruan", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTwo very recent landmark large-scale studies show that loss-of-function mutations in APOC3 are associated with lower levels of plasma triglycerides, and carriers of these mutations have a reduced risk of coronary heart disease in European and African individuals. Tang et al. performed a timely replication study in 4,470 Chinese individuals. Unexpectedly, no loss-of-function mutations identified in the European and African populations were found in the Chinese population. This important study not only highlights the difference in genetic susceptibility to cardiovascular disease in different ethnic populations, but also suggests that APOC3 variants are not applicable to the Chinese population to predict risk for ischemic vascular disease.APOC3 may still be an important regulator of lipid metabolism in Chinese, and novel variants of this gene remain to be identified in this ethnic population. Consequently, the authors need to change their conclusion from “Therefore, APOC3 may not be an ideal predictor for risk of ischemic vascular disease in the Chinese population” to “Therefore, APOC3 variants identified in the European and African population may not be an ideal predictor for risk of ischemic vascular disease in the Chinese population”.", "responses": [] }, { "id": "6866", "date": "10 Dec 2014", "name": "Bin Zhang", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors performed a confirmatory study for the recently reported protective effects of loss-of-function mutations in APOC3 in the Chinese population. Not a single loss-of-function mutation was identified in exons and exon-intron junctions of APOC3 in a total of 4470 study subjects. The authors concluded that APOC3 is not a good predictor of risk of ischemic vascular disease in the Chinese population.This is a worthwhile study and the information, although negative, is certainly useful. However, based on the data reported in the paper, it is premature to conclude that functional variants in APOC3 are very rare and/or not related to ischemic vascular disease in the Chinese population. The data merely conclude that detrimental mutations are very rare in the sequenced regions of APOC3 gene in the Chinese population. Potential effects from large deletions of the gene and mutations in regulatory regions of the gene cannot be excluded. In fact, there have been reports of promoter polymorphisms that affect the expression of APOC3 (ref. 28-31 in Jørgensen et. al). Unless additional, more comprehensive mutational studies are performed in the cohort, conclusions should be modified to reflect the limitations of the current study.There have been examples of synonymous mutations that affect the translation of mRNA and therefore affect the protein level. Also, intronic mutations distant from splice sites could affect splicing. Authors should clearly state reasons why they believe that the variants identified in the study have no functional consequences.References should be added to the end of the second sentence of the paper (while only “a” few studies have identified protective variants conferring reduced risk).", "responses": [] } ]	1	https://f1000research.com/articles/3-270
https://f1000research.com/articles/3-312/v1	22 Dec 14	{ "type": "Research Article", "title": "Teaching basic numeracy, predictive models and socioeconomics to marine ecologists through Bayesian belief networks", "authors": [ "Richard Stafford", "Rachel Williams", "Rachel Williams" ], "abstract": "Teaching numeric disciplines to higher education students in many life sciences disciplines is highly challenging. In this study, we test whether an approach linking field observations with predictive models can be useful in allowing students to understand basic numeracy and probability, as well as developing skills in modelling, understanding species interactions and even community/ecosystem-service interactions. We presented a field-based lecture in a morning session (on rocky shore ecology), followed by an afternoon session parameterising a belief network using a simple, user-friendly interface. The study was conducted with students during their second week of a foundation degree, hence having little prior knowledge of these systems or models. All students could create realistic predictive models of competition, predation and grazing, although most initially failed to account for trophic cascade effects in parameterising their models of the rocky shore they had previously seen. The belief network was then modified to account for a marine ecosystem management approach, where fishing effort and economic benefit of fishing were linked to population abundance of different species, and management goals were included. Students had little difficultly in applying conceptual links between species and ecosystem services in the same manner as between species. Students evaluated their understanding of a range of variables from rocky shore knowledge to marine management as increasing over the session, but the role of the predictive modelling task was indicated as a major source of learning, even for topics we thought may be better learned in the field. The study adds evidence to the theories that students benefit from exposure to numeric topics, even very early in their degree programmes, but students grasp concepts better when applied to real world situations which they have experience of, or perceive as important.", "keywords": [ "Bayesian belief networks", "higher education", "marine biology", "rocky shore", "predictive models" ], "content": "Introduction\n\nWhile there are considerable differences between students’ understanding and academic strengths worldwide, within much of the native English speaking world large proportions of students studying life science disciplines at higher education level have weaknesses with numeracy, and associated topics, such as statistics (Feser et al., 2013). In many cases, ‘fears’ of mathematics have arisen and continued throughout their schooling, however, avoidance of numeric topics is not possible in many life science disciplines, especially in subjects such as ecology, where statistics form an important part of any independent research (e.g. Chalmers & Parker, 1989).\n\nMany case studies have demonstrated that quantitative skills in ecology can be better taught if taught in a subject context (i.e. analysing data the students have collected in field based studies – Bäumer, 1999; Nolan & Speed, 1999; Yilmaz, 1996). Despite this, many students remain unconfident of quantitative elements of their courses, and few have the confidence apply even simple mathematics outside the confines of a few simple statistical tests (Grainger, 2010; Tariq, 2002; Tariq, 2004).\n\nA possible solution to increase quantitative skills and confidence in students is the use of Bayesian belief networks. In their simplest form, Bayesian belief networks are a method of formalising uncertainty or probability from a number of events (Grover, 2013). From an ecological community perspective, this could amount to the calculated probability of a species decreasing in abundance, but based on known increases in both food supply and predation risk (Hammond & Ellis, 2002; Stafford et al., 2013). However, traditional Bayesian belief networks do not cope well with reciprocal interactions (such as competition between two species), and revised methods using some aspects of Bayesian inference have been developed (Stafford R. and Gardner E. unpublished data). These revised methods also have a simple, user-friendly interface. They use Microsoft Excel as an interface, with a hidden VBA script performing the calculations. As such, the software used is familiar to the students, rather than complex modelling environments or coding platforms often used for predictive models.\n\nGiven the identified need for quantitative skills in graduates, and even in post-doctoral researchers (Grainger, 2010), the aim of this study was determine if it was possible to introduce predictive modelling to students at an early point in their education, and whether this predictive modelling would benefit student learning about ecology in general, rather than being seen as extra, and unnecessary, complexity in learning key concepts. To do this, we combined a field visit to a rocky shore, followed by an afternoon using a predictive belief network, initially based on their knowledge of the rocky shore. We quantified the success of the technique both through student questionnaires and through examining the solutions they provided to problems requiring the use of the predictive networks.\n\n\nMethods\n\nThe study group consisted of 12 students enrolled on a foundation degree (UK level 4) in Marine Ecology. The students were based in an educational college normally specialising in further education (pre-18 years old), but with some higher education (university level or post-18 courses) offered. The marine ecology course formed part of the higher education provision.\n\nStudents were in their second week of university level education, and from mixed backgrounds (~1/3rd mature students - 3 or more years after finishing school, and 2/3rds recent school or college leavers), however, none had previously formally studied ecology or ecological interactions beyond that covered in a typical school syllabus (UK A-levels or equivalent).\n\nThe field teaching session took place at Osmington Mills in Dorset, UK in early October 2014. Teaching consisted of an introduction lecture to rocky shore ecology, particularly examining ideas relating to vertical distribution of organisms (zonation) and exploring why small scale spatial patterns may have occurred (i.e. patches of green algae on the high shore, patches of barnacles). This gave a comprehensive introduction to physical limitations on organisms, as well as biological interactions (particularly interspecific competition and tropic interactions). Specifically eight key organisms were identified (Fucoid algae – Fucus spp., green algae – Enteromorpha spp., coralline algae - Corallina officinalis, barnacles – Chthalamalus and Semibalanus, dogwhelks – Nucella lapillus, periwinkles – Littorina littorea, topshells – Osilinus lineatus, and limpets Patella spp.), and after a 90 min field lecture, students were told to look for these species and consider their ecological interactions with each other (the lecture had given the basic ‘role’ of each species – i.e. grazer, filter feeder, predator; but had not provided details of interactions between the species). These eight organisms were all common on the mid shore, and were the species incorporated in the predictive model.\n\nThe full details of the belief network model used are provided as appendices (Supplementary File 1), as well as in VBA code within the downloadable Microsoft Excel files (Supplementary File 2). It comprised three ‘worksheets’ in a Microsoft Excel ‘workbook’. One worksheet asked for an indication of which species were directly interacting (i.e. competitively, trophically, mutualistically), and simply followed a grid system whereby a species in a column on the grid is effected by a species in the row on the grid. This worksheet automatically updates a second worksheet, indicating where ‘probabilities’ of species interactions need to be filled in. The top table on this second worksheet asks for a number between 0 and 1 indicating the probability of a target species increasing, assuming the probability of the ‘causing’ species is increasing. Largely, once this table is completed, typically with 15–20 different interaction probabilities (depending on how many interactions are specified on the previous worksheet), the model is complete. However, it is possible to specify non-reciprocal relationships if desired (for example, the probability of barnacles increasing, given an increase in predatory dogwhelks could be low (e.g. p = 0.1). The spreadsheet would update automatically to indicate a decrease in barnacles, if dogwhelks increased would be the reciprocal of this (p = 0.9), although these values could be manually altered if preferred. In this case, no students altered the other probability values.\n\nThe final worksheet contained the ‘prior’ probabilities of species changes. In this case, two scenarios were given to predict community level changes. Firstly, increases in dogwhelks, and secondly increases in periwinkles. These were described in the manner of actual experiments conducted at Osmington Mills (manipulation experiments designed to increase the abundance of these species – data set provided as Dataset 1). In these belief networks, only a prior probability of directly affected species should be altered, with the remainder of probabilities for species increases or decreases remaining at 0.5 for both increase and decrease. In the specific task set, students were asked to consider interactions over a three to four week period, partly to compare with experimental data that was collected over this time frame, and partly to simplify bottom up relationships (starvation is likely to take much longer than this in typical intertidal molluscs). Equally, while slow growing seaweeds will be unlikely to exhibit growth and therefore increased percentage cover, rapid growing green algae will be able to increase in abundance (Lubchenco, 1978).\n\nPressing a ‘calculate’ button then runs the VBA code and calculates posterior probabilities, indicating the likelihood of species increasing or decreasing. Results were then discussed in small groups, and where errors were made, the reasons for these errors were discussed.\n\nA marine management scenario was then conducted, using the same spreadsheet template and underlying VBA code. A scenario based upon common commercial fish and shellfish (cod, haddock, whiting, sole and scallops) as well as important biotic habitat types (sea fans) was given. This time, however, trawling effort and fisher income were two non-biological interactions (Supplementary File 3). Students were asked complete the network in the same way, just considering what species or service was effected by others, and then parameterising the network with probabilities, in the same manner as before. In this case, they were asked to explore options for enhancing sea fan populations, without reducing fisher income.\n\nAn anonymous questionnaire was provided to each student, asking how much they had learned about various topics during the day (from rocky shore ecology through to understanding probability and uncertainty – Figure 1; Supplementary File 4). In addition to ranking each on a 1–10 scale, they were asked to provide an answer as to where they thought they had learned most about the topic (either during the field lecture, or the afternoon computer session). Again, this was on a 1–10 scale, where 1 was only from the morning field session, 5 was equally from both sessions, and 10 was only from the afternoon session. The collection of these data from students was approved by the Science, Technology and Health ethics committee of Bournemouth University.\n\nFor each learning outcome examined, the median score was 7. Contribution was calculated using the equations C1 = M1*((10-M2)/10) and C2 = 10-C1, where C1 is the contribution made by field-based lecture, M1 is the median value of the students’ response to how much they learned about the topic (in this case, all equal to 7) and M2 is the median value of the students’ response to how much they learned from the different teaching methods (1 indicating fully from the field-based lecture and 10 indicating fully from the computer-based practical).\n\n\nResults\n\nAfter a few questions regarding ‘what to do’, students (working in pairs) all produced sensible interaction links in the model. Typical initial questions involved which worksheets to fill in and a general reluctance to ‘break’ the software by doing something wrong. Filling in the probability values resulted in a minor problem of students not considering exactly what they were trying to predict (the probability of a species increasing, given that another species had increased – in this example, where most species interactions are negative, it may be easier to parameterise the model as probability of a species decreasing, given another had increased). Four of the six groups needed this re-explaining, however, once this was pointed out and otherwise independently, all groups managed to produce working belief network simulations which accurately predicted real results from previously conducted experiments. The only area where the majority of groups (five out of six) struggled was in complex, indirect interactions, which only occurred as a result of a tropic cascade. Most groups failed to predict the competitive interactions for space between seaweed species, despite correctly predicting the tropic relationships between grazers and green algae, but other than this one interaction, results were almost identical to the ‘expert’ parameterised networks created by the authors, and produced similar values to real experiments conducted on rocky shores (see Dataset 1 for real experimental data).\n\nStudent evaluations showed that students felt they had learned significant amounts about each topic, with median scores of 7 for all questions (with 25% and 75% quartiles between 6.5 and 9 in all cases). Knowledge of rocky shore ecology and ecological interactions was ranked as equally learned between the field and computer sessions (both with median values of 5) and knowledge of marine management and probability was greater in the computer based sessions (median scores of 7 and 8 respectively; Figure 1). In all cases, the full IQR was 2 or less indicating the majority of students scored in a similar manner (Dataset 2 provides the full data for each student – note, as the survey was optional, only 11 of the 12 students present completed this).\n\n\nDiscussion\n\nThis study indicates that students (even at a very early stage in their academic journeys) are capable of understanding and applying quantitative techniques if presented in a logical and intuitive way. Furthermore, it demonstrates that their ability to learn these techniques can be integrated with development of ecological knowledge and understanding; acting not as a hindrance to, but as a compliment to the understanding of key concepts.\n\nMuch previous research has demonstrated that quantitative methods need to be taught in the context of the academic discipline (Bäumer, 1999; Nolan & Speed, 1999; Yilmaz, 1996). However, this approach can often seem rather forced to students, especially early in their studies. Examples are often based around simple data collecting exercises rather than conceptual issues or real research projects. Although students are capable of performing statistical tests, for example, on a specific dataset at the time, they find it difficult to remember and transfer the knowledge to a novel situation (Chance, 2002). However, embedding more quantitative elements regularly to courses to enhance learning of key concepts may result in less fear being associated with dealing with numbers, and a greater ability to synthesise more quantitative information such as inferential statistics when required.\n\nThe role of technology in education is becoming a prominent issue, with a number of case studies demonstrating how it can be useful in the classroom and even during field work (France & Welsh, 2012; Webb & Stafford, 2013; Welsh & France, 2012). Simulations have also been used to teach complex ecological concepts such as experimental design in place of (or in addition to) field or laboratory based studies (Stafford et al., 2010). With on-going improvements to technology (e.g. development of specific apps for tablet computers), it may be possible to use spreadsheet models such as this directly in the field. An advantage of such an approach would be that the simulation app could be combined with field guides and information on the species being studied, potentially allowing a more fully immersive learning experience.\n\nThe simplicity of use of belief networks, especially when combined with a familiar user interface, was clearly demonstrated here, with students being able to make, parameterise and run simulations of ecological systems and coupled socio-ecological systems. These results add further weight to the intuitiveness of the approach. Combined with uncertainty in detailed knowledge of many marine ecosystems (in terms of exact population sizes, recruitment and species interactions - Hilborn & Walters, 1992; Magnusson, 1995; Pope, 1991), and the need to adhere to what could be perceived as ‘crude’ policy measures (e.g. no decrease in the population size of a certain species – DEFRA, 2012), models such as these used in this study would fit well with the demands and requirements of policy makers, providing sufficient detail for achieving policy goals with limited data and in an intuitive manner.\n\n\nData availability\n\nF1000Research: Dataset 1. Data from manipulative experiment to assess community effects of dogwhelk manipulation (addition of 10 dogwhelks to each of 5 × ~1.5 × 1.5 × 1.5 m boulders) or periwinkle manipulation (addition of 10 periwinkles to 5 different, but similar sized boulders)., 10.5256/f1000research.5981.d41189 (Stafford & Williams, 2014a).\n\nF1000Research: Dataset 2. Raw data values of student responses to surveys, 10.5256/f1000research.5981.d41190 (Stafford & Williams, 2014b).", "appendix": "Author contributions\n\n\n\nRS wrote the code for the Bayesian belief model. RS and RLW both conceived the study, conducted the taught sessions and collected, analysed the results and contributed to writing the paper. Both authors have agreed the content of the final draft of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nSupplementary information\n\nSupplementary File 1: Mathematical details and description of the belief network model.\n\nClick here to access the data.\n\nSupplementary File 2: Microsoft Excel file (with VBA script and macros) realising the belief network model for the rocky shore scenario.\n\nThe file is parameterised as per authors’ values, but is fully editable to allow students to alter these.\n\nClick here to access the data.\n\nSupplementary File 3: Microsoft Excel file (with VBA script and macros) realising the belief network model for the marine management scenario.\n\nThe file is parameterised as per authors’ values, but is fully editable to allow students to alter these.\n\nClick here to access the data.\n\nSupplementary File 4: Copy of survey provided to students to determine what they considered they learned, and whether they learned it from field lectures or computer based practical classes.\n\nNote – survey was explained orally to students before completing it.\n\nClick here to access the data.\n\n\nReferences\n\nBäumer HP: Teaching Multivariate Data Analysis in the Fields of Biology and Ecology. Proceedings of the International Statistics Instituted 52nd Session, Helsinki, Finland, 1999. Reference Source\n\nChalmers N, Parker P: The OU Project Guide: Fieldwork and Statistics for Ecological Projects. Field Studies Council: Shrewsbury, UK, 1989. Reference Source\n\nChance BL: Components of Statistical Thinking and Implications for Instruction and Assessment. J Stat Educ. 2002; 10(3). Reference Source\n\nDEFRA: Marine Strategy Part One: UK Initial Assessment and Good Environmental Status. DEFRA, London, UK, 2012. Reference Source\n\nFeser J, Vasaly H, Herrera J: On the edge of mathematics and biology integration: improving quantitative skills in undergraduate biology education. CBE Life Sci Educ. 2013; 12(2): 124–128. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFrance D, Welsh K: The Future of Higher Education Fieldwork in Geography, Earth and Environmental Sciences. Higher Education Academy: York, 2012. Reference Source\n\nGrainger K: Review of the Skills Needs in the Environment Sector. Natural Environmental Research Council: Swindon, 2010. Reference Source\n\nGrover J: Strategic economic decision-making: using Bayesian networks to solve complex problems. Springer: New York, 2013. Publisher Full Text\n\nHammond TR, Ellis JR: A meta-assessment for elasmobranchs based on dietary data and Bayesian networks. Ecol Indic. 2002; 1(3): 197–211. Publisher Full Text\n\nHilborn R, Walters CJ: Quantitative Fisheries Stock Assessment: Choice, Dynamics and Uncertainty. Chapman and Hall: New York, 1992. Reference Source\n\nLubchenco J: Plant species diversity in a marine intertidal community: importance of herbivore food preference and algal competitive abilities. Am Nat. 1978; 12(983): 23–39. Publisher Full Text\n\nMagnusson KG: An overview of the multispecies VPA—theory and applications. Reviews in Fish Biology and Fisheries. 1995; 5(2): 195–212. Publisher Full Text\n\nNolan D, Speed TP: Teaching statistics theory through applications. Am Statistician. 1999; 53(4): 370–375. Publisher Full Text\n\nPope JG: The ICES Multispecies Assessment Working Group: Evolution, Insights, and Future Problems. ICES mar Sei Symp. 1991; 193: 22–33. Reference Source\n\nStafford R, Goodenough AE, Davies MS: Assessing the effectiveness of a computer simulation for teaching ecological experimental design. Biosci Edu. 2010; 15: 1–9. Publisher Full Text\n\nStafford R, Smith VA, Husmeier D, et al.: Predicting ecological regime shift under climate change: new modelling techniques and potential of molecular-based approaches. Curr Zool. 2013; 59(3): 403–417. Reference Source\n\nStafford R, Williams RL: Dataset 1. Data from manipulative experiment to assess community effects of dogwhelk manipulation (addition of 10 dogwhelks to each of 5 × ~1.5 × 1.5 × 1.5 m boulders) or periwinkle manipulation (addition of 10 periwinkles to 5 different, but similar sized boulders). F1000Research. 2014a. Data Source\n\nStafford R, Williams RL: Dataset 2. Raw data values of student responses to surveys. F1000Research. 2014b. Data Source\n\nTariq VN: A decline in numeracy skills among bioscience under graduates. J Biol Educ. 2002; 36(2): 76–83. Publisher Full Text\n\nTariq V: Numeracy, mathematical literacy and the life sciences. MSOR Connections. 2004; 4(2): 25–29. Publisher Full Text\n\nWebb J, Stafford R: Location-based mobile phone applications for increasing student engagement with field-based extra-curricular activities. Planet. 2013; 27(1): 29–34. Publisher Full Text\n\nWelsh K, France D: Spotlight on….Smartphones and fieldwork. Geography. 2012; 97(1): 47–51. Reference Source\n\nYilmaz MR: The Challenge of Teaching Statistics to Non-Specialists. J Stat Educ. 1996; 4(1). Reference Source" }	[ { "id": "7314", "date": "14 Jan 2015", "name": "Vivien Sieber", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting and relevant study on a topic that is, increasingly, recognized as important. The authors restrict their discussion to Life Sciences, in particular marine ecology whilst this study is potentially of wider interest to the scientific community. The authors may find some of the included references helpful in expanding their argument.Assuming this paper is interesting to a wider community rather than restricted to specialist marine ecologists; it would be helpful to give a brief definition of Bayesian belief networks.MethodsPlease give the HE qualification/s participants were working towards to facilitate comparison with other studies.Predictive model/Belief networkThis text would benefit from clarification as it is rather difficult to follow for non-specialists. Might a diagram or flow chart help?Sentence 3: ‘effected’ should be ‘affected’SurveyIt would be conventional to insert the rubric once at the top of the questionnaire rather than repeating it in every question. Respondents might be discouraged by this additional burden.ResultsDeveloping new approaches to teaching are always difficult as teaching is constrained by practicalities such as timetabling. It would be interesting if it were practically possible to compare outcomes after switching the field-work element with the simulation.It would be easier for the reader if the responses to the survey were summarized and presented graphically.Remove the link to Supplementary file 4 0c718f4f-6cb7-4030-823c-94f3687abe47.docx from the legend Dataset 2 to avoid confusion.DiscussionAgain, the discussion might usefully be expanded to include a wider range of subject fields, perhaps including evidence supporting the role of simulations in generating understanding. The authors might wish to comment on the difference between the ability to carry out a calculation and the skills needed to interpret data.", "responses": [] }, { "id": "8317", "date": "15 Apr 2015", "name": "Vicki N. Tariq", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nA useful contribution to strategies aimed at helping life sciences undergraduates appreciate the importance of quantitative skills, develop their own quantitative skills and (hopefully) become less anxious about mathematics and statistics within the first few weeks of their transition onto a higher education programme. Introduction2nd paragraph: Suggest changing to “in ecology can be better learned/developed if they are taught in a subject context ……” Also ….. “and few have the confidence to apply…..”3rd paragraph: A simple diagram might help readers understand Bayesian belief networks in an ecological context.4th paragraph: “….. the aim of this study was to determine …..”; “….. seen as an extra, and unnecessary complexity…..”; “….. we combined a field visit to a rocky shore with an afternoon using …..”. Methods1st paragraph: “The students were based in a college normally specialising in further education (pre-18 years old), but offering some higher education courses.”Predictive model/Belief network: requires more clarification for non-specialists. It might also help if the three individual worksheets in Supplementary File 2 were numbered (and referred to by these numbers in the text) and each worksheet contained more explanation so that the reader wasn’t trying to move back and forth between the manuscript and the Excel worksheets. Also in sentence 3 – “effected” should be “affected”.Marine management scenario: 4th sentence – “Students were asked to complete ……”; “effected” should be “affected”.Student evaluation of learning: the rubric in the questionnaire should have been presented only once at the beginning – its repetition before each question is tedious (to say the least) to students and readers of this article alike. ResultsFigure 1: the horizontal axis needs to be labelled as ‘Contribution’The two references in the text to “tropic cascade” should be “trophic cascade”The authors state that “Student evaluations showed that students felt they had learned significant amounts about each topic …..”, “students (working in pairs) all produced sensible interaction links in the model”, and “[they produced] working belief network simulations which accurately predicted real results from previously conducted experiments”, but did the authors use any other means to assess each individual student’s quantitative skills (e.g. their understanding of probability) and knowledge of rocky shore ecology (e.g. species interactions, marine management)? – the students, particularly when working in pairs, may have over-estimated the extent of their learning, in which case their self-evaluation of their knowledge and competence may not have matched their actual knowledge and competence? The authors may have needed to do more than “[quantify] the success of the technique ………. through examining the solutions [the pairs of students] provided to problems requiring the use of predictive networks.” (final sentence of the Introduction). Discussion2nd paragraph, final sentence: “However, embedding more quantitative elements regularly in courses to enhance ….”The discussion would benefit from the authors addressing the points raised by Vivien Sieber.", "responses": [] } ]	1	https://f1000research.com/articles/3-312
https://f1000research.com/articles/3-310/v1	19 Dec 14	{ "type": "Case Report", "title": "Case Report: Urgent endovascular treatment of subclavian artery injury after blunt trauma", "authors": [ "Taka-aki Nakada", "Koji Idoguchi", "Hiroshi Fukuma", "Hidefumi Ono", "Shota Nakao", "Tetsuya Matsuoka", "Koji Idoguchi", "Hiroshi Fukuma", "Hidefumi Ono", "Shota Nakao", "Tetsuya Matsuoka" ], "abstract": "Subclavian arterial injury is rare and potentially life-threatening, particularly when it leads to arterial occlusion, causing limb ischemia, retrograde thromboembolization and cerebral infarction within hours after injury. Here we report a blunt trauma case with subclavian arterial injury, upper extremity ischemia, and the need for urgent treatment to salvage the limb and prevent cerebral infarction. A 41-year-old man had a left, open, mid-shaft clavicle fracture and left subclavian artery injury accompanied by a weak pulse in the left radial artery, decreased blood pressure of the left arm compared to the right, and left hand numbness. Urgent debridement and irrigation of the open clavicle fracture was followed by angiography for the subclavian artery injury. The left distal subclavian artery had a segmental dissection with a thrombus. Urgent endovascular treatment using a self-expanding nitinol stent successfully restored the blood flow and blood pressure to the left upper extremity. Endovascular treatment is a viable option for cases of subclavian artery injury where there is a risk of extremity ischemia and cerebral infarction.", "keywords": [ "Endovascular treatment", "blunt trauma", "subclavian artery injury", "open clavicle fracture" ], "content": "Introduction\n\nSubclavian arterial injury caused by blunt trauma is rare with potentially high morbidity and mortality1,2. Open clavicle fractures caused by blunt trauma are also rare3,4. Here we report a blunt trauma case with open clavicle fractures and subclavian artery injury accompanied by upper extremity ischemia and the need for urgent treatment.\n\n\nCase report\n\nA 41-year-old man, who had no significant previous medical or family history, was thrown from the rear seat of a vehicle during an accident on the motorway. He was transferred to the emergency department of our hospital. Upon admission, he had an open airway, normal breathing with a respiratory rate of 16 breaths/min, was hemodynamically stable with a blood pressure of 123/79 mmHg, and a pulse rate of 88 beats/min. He was conscious and scored E3 for eye opening, V5 for verbal response, and M6 for motor response on the Glasgow Coma Scale. He had a left pneumothorax, a left, open, mid-shaft clavicle fracture accompanied by a 10 mm-sized laceration with numerous subcutaneous air bubbles trapped in the soft tissue on the lateral end of the clavicle, and left subclavian arterial injury (Gustilo Grade I) (Figure 1A–C). He had multiple lacerations of the forehead without abnormal findings in computed tomography of the head and neck. Both hands were warm with brisk capillary refill in the fingers. The radial and ulnar pulses in the left hand were palpable, but markedly weaker compared to those of the right hand. The blood pressure of the left arm was approximately half that of the right arm blood pressure. Despite no muscle weakness in the upper extremities, the patient had left hand numbness. The Injury Severity Score was 11. The patient was treated with urgent debridement and irrigation for the open clavicle fracture in the operating room followed by urgent angiography for the subclavian artery injury. Initial selective angiography of the left subclavian artery via the right common femoral artery revealed a segmental dissection of the distal subclavian artery with preserved blood flow to the left upper extremity (Figure 2A). Subsequent intravascular ultrasound via the left brachial artery revealed an intimal flap and a compressed true lumen by a thrombus of the pseudo lumen in the distal subclavian artery (length of the lesion, 3 cm). An 8 mm × 40 mm self-expanding nitinol stent (Smart Control, Cordis) was deployed. Adequate stent expansion and restoration of blood flow of the subclavian artery were confirmed (Figure 2B). After the endovascular stenting, the left radial and ulnar pulses were remarkably improved and the blood pressure difference between the left and right arm was significantly eliminated. Antithrombotic therapy to prevent stent thrombosis using intravenous heparin targeting aPTT of 2 times the control aPTT for 9 days was followed by an antiplatelet therapy using aspirin 100 mg plus cilostazol 200 mg daily for 12 months. On day 6, an open reduction and internal fixation of the clavicle fracture using a Kirschner wire were performed. The patient was discharged on day 22 and continued to be free of complications at the 2-month follow-up with stent patency determined using color duplex ultrasonography.\n\n(A) Subclavian artery injury shown on contrast-enhanced computed tomography. (B and C) Clavicle fractures and subclavian artery injury shown on three-dimensional computed tomography angiography.\n\n(A) Angiography showing segmental dissection of the distal subclavian artery with preserved blood flow to the left upper extremity. (B) Angiography of the subclavian artery showing an adequate stent expansion and restoration of blood flow.\n\n\nDiscussion\n\nClavicle fractures are common injuries and mostly treated non-operatively with good outcomes, while open clavicle fractures due to blunt trauma are rare, accounting for 0.2–1.3% of all clavicle fractures in a trauma clinic or Level I trauma center3,4. Open clavicle fractures caused by penetrating trauma are frequently associated with a great vessel injury, including subclavian artery injury, compared to those caused by blunt trauma3.\n\nSubclavian artery injuries through blunt trauma are rare with a reported incidence of less than 1% of all arterial injuries or thoracic traumatic injuries5–7. Subclavian artery injuries are caused by stretching, transection, or compression of the subclavian artery by broken bone fragments. Unexpected neurovascular symptoms, a pseudoaneurysm rupture, or a thrombus associated with upper extremity ischemia often initiate weeks or months after initial injury. There have been reports of patients who had delayed symptom recognition but were treated successfully in late phases8,9. However, there have been cases with massive hemorrhage due to transection of the subclavian artery1 or cerebral infarction due to occlusion of the subclavian artery2 within hours after injury, highlighting the importance of urgent therapeutic management of subclavian artery injury. Our case presented an intimal injury of the subclavian artery with a thrombus leading to upper extremity ischemia, which could cause retrograde thromboembolization and cerebral infarction. We thus urgently treated for prevention of cerebral infarction and to salvage the limb.\n\nAn open surgical approach is one treatment option for subclavian artery injury. However, this approach requires an extensive incision to obtain proximal and distal control, which is invasive, difficult to perform, and associated with high morbidity6,10. In our case, the patient had an open fracture, which is a risk factor for graft infection in vascular surgery. Advances in endovascular treatments for vascular injuries have achieved increasing success for treatment of subclavian artery injury caused by penetrating trauma such as a gunshot, stab, or iatrogenic catheter injury10. Endovascular treatment is a viable option for cases of subclavian artery injury where there is a risk of extremity ischemia and cerebral infarction.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and images was obtained from the patient.", "appendix": "Author contributions\n\n\n\nTN prepared the first draft of the manuscript. KI, HF, HO, SN and TM provided additional editing and expert content. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nKendall KM, Burton JH, Cushing B: Fatal subclavian artery transection from isolated clavicle fracture. J Trauma. 2000; 48(2): 316–318. PubMed Abstract \| Publisher Full Text\n\nChavali S, Shukla U, Chauta S: Traumatic subclavian arterial thrombosis presenting with cerebral infarct--a case report. Heart Lung Circ. 2014; 23(10): e202–206. PubMed Abstract \| Publisher Full Text\n\nGottschalk HP, Dumont G, Khanani S, et al.: Open clavicle fractures: patterns of trauma and associated injuries. J Orthop Trauma. 2012; 26(2): 107–109. PubMed Abstract \| Publisher Full Text\n\nRobinson CM: Fractures of the clavicle in the adult. Epidemiology and classification. J Bone Joint Surg Br. 1998; 80(3): 476–484. PubMed Abstract\n\nFranz RW: Delayed treatment of a traumatic left subclavian artery pseudoaneurysm. Vasc Endovascular Surg. 2008; 42(5): 482–485. PubMed Abstract \| Publisher Full Text\n\nAssenza M, Centonze L, Valesini L, et al.: Traumatic subclavian arterial rupture: a case report and review of literature. World J Emerg Surg. 2012; 7(1): 18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTomaszek DE: Combined subclavian artery and brachial plexus injuries from blunt upper-extremity trauma. J Trauma. 1984; 24(2): 161–163. PubMed Abstract \| Publisher Full Text\n\nMandal AK, Jordaan J, Missouris CG: Fractured clavicle and vascular complications. Emerg Med J. 2004; 21(5): 648. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGullo J, Singletary EM, Larese S: Emergency bedside sonographic diagnosis of subclavian artery pseudoaneurysm with brachial plexopathy after clavicle fracture. Ann Emerg Med. 2013; 61(2): 204–206. PubMed Abstract \| Publisher Full Text\n\nCarrick MM, Morrison CA, Pham HQ, et al.: Modern management of traumatic subclavian artery injuries: a single institution’s experience in the evolution of endovascular repair. Am J Surg. 2010; 199(1): 28–34. PubMed Abstract \| Publisher Full Text" }	[ { "id": "7100", "date": "14 Jan 2015", "name": "Urvi Shukla", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFirst of all, let me congratulate the team on a case report that is well written and a patient that was well managed. This report again highlights the fact that displaced clavicular fractures can lead to vascular complications, either thrombotic or bleed. These complications may lead to greater morbidity or even a life threatening event. It needs a high index of suspicion to recognize these injuries, more so because timely intervention may save someone's limb or life. The authors have recognized the injury early and managed it aptly. Endovascular repair of vessel injury is now standard of care with sophisticated stents and procedural advances. Subclavian artery dissection with thrombosis in the false lumen has been managed as per evidence base.", "responses": [] }, { "id": "7279", "date": "14 Jan 2015", "name": "Tal Hörer", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI would like to congratulate the authors for the successful treatment and report. This is a report about endovascular trauma treatment and in my eyes, state of the art for modern trauma surgery.I would humbly ask that the authors look at the English as there are some minor English corrections that can be done.", "responses": [] } ]	1	https://f1000research.com/articles/3-310
https://f1000research.com/articles/3-115/v1	21 May 14	{ "type": "Research Article", "title": "Systems analysis of the prostate tumor suppressor NKX3.1 supports roles in DNA repair and luminal cell differentiation", "authors": [ "Chih-Cheng Yang", "Alicia Chung", "Chia-Yu Ku", "Laurence M. Brill", "Roy Williams", "Dieter A. Wolf", "Alicia Chung", "Chia-Yu Ku", "Laurence M. Brill", "Roy Williams" ], "abstract": "NKX3.1 is a homeobox transcription factor whose function as a prostate tumor suppressor remains insufficiently understood because neither the transcriptional program governed by NKX3.1, nor its interacting proteins have been fully revealed. Using affinity purification and mass spectrometry, we have established an extensive NKX3.1 interactome which contains the DNA repair proteins Ku70, Ku80, and PARP, thus providing a molecular underpinning to previous reports implicating NKX3.1 in DNA repair. Transcriptomic profiling of NKX3.1-negative prostate epithelial cells acutely expressing NKX3.1 revealed a rapid and complex response that is a near mirror image of the gene expression signature of human prostatic intraepithelial neoplasia (PIN). Pathway and network analyses suggested that NKX3.1 actuates a cellular reprogramming toward luminal cell differentiation characterized by suppression of pro-oncogenic c-MYC and interferon-STAT signaling and activation of tumor suppressor pathways. Consistently, ectopic expression of NKX3.1 conferred a growth arrest depending on TNFα and JNK signaling. We propose that the tumor suppressor function of NKX3.1 entails a transcriptional program that maintains the differentiation state of secretory luminal cells and that disruption of NKX3.1 contributes to prostate tumorigenesis by permitting luminal cell de-differentiation potentially augmented by defects in DNA repair.", "keywords": [ "NKX3.1 encodes a homeodomain transcription factor whose expression is largely restricted to the prostate and controlled by androgen. The gene is located on chromosome 8p21 in a region frequently deleted in early prostate cancers (reviewed in1", "2). Studies in Nkx3.1 knockout mice have provided compelling evidence that Nkx3.1 is a prostate tumor suppressor3–5. These mice develop prostatic intraepithelial neoplasia (PIN)", "a precancerous lesion characterized by hyperproliferation of dysplastic cells", "indicating that Nkx3.1 is haploinsufficient for PIN suppression6. Additional studies showed that serial passage of PIN-like lesions from Nkx3.1 mutant mice can undergo progressively severe histopathological alterations5. Finally", "loss of Nkx3.1 can cooperate with loss of Pten and p27 in prostate cancer development in mice7", "8", "while Nkx3.1 overexpression inhibits cell proliferation in Pten null epithelial grafts9. These data indicate that the diminished expression of NKX3.1 that is frequently observed in human prostate cancers10 is involved in the initial stage of prostate carcinogenesis. While the tumor suppressor function of NKX3.1 remains poorly defined at the molecular level", "the knockout phenotypes suggested that Nkx3.1 controls genes involved in prostate development", "differentiation", "and maintenance of tissue integrity." ], "content": "Introduction\n\nNKX3.1 encodes a homeodomain transcription factor whose expression is largely restricted to the prostate and controlled by androgen. The gene is located on chromosome 8p21 in a region frequently deleted in early prostate cancers (reviewed in1,2). Studies in Nkx3.1 knockout mice have provided compelling evidence that Nkx3.1 is a prostate tumor suppressor3–5. These mice develop prostatic intraepithelial neoplasia (PIN), a precancerous lesion characterized by hyperproliferation of dysplastic cells, indicating that Nkx3.1 is haploinsufficient for PIN suppression6. Additional studies showed that serial passage of PIN-like lesions from Nkx3.1 mutant mice can undergo progressively severe histopathological alterations5. Finally, loss of Nkx3.1 can cooperate with loss of Pten and p27 in prostate cancer development in mice7,8, while Nkx3.1 overexpression inhibits cell proliferation in Pten null epithelial grafts9. These data indicate that the diminished expression of NKX3.1 that is frequently observed in human prostate cancers10 is involved in the initial stage of prostate carcinogenesis. While the tumor suppressor function of NKX3.1 remains poorly defined at the molecular level, the knockout phenotypes suggested that Nkx3.1 controls genes involved in prostate development, differentiation, and maintenance of tissue integrity.\n\nLike other NKX class homeoproteins, NKX3.1 can function as a transcriptional repressor by binding a non-canonical homeodomain DNA motif such as naturally occurring in the mouse androgen receptor promoter9 or artificially presented in synthetic reporter genes11. Transcriptional repression may involve NKX3.1-mediated recruitment of co-repressors12 and the histone deacetylase, HDAC19. A second mode of trans-repression found for the prostate-specific antigen (PSA) gene occurs independently of NKX3.1 promoter binding sites, but through repressive interaction with transcriptional activators such as SP113 and prostate-derived ETS factor (PDEF14). NKX3.1 was also shown to activate gene transcription, either through direct promoter binding as in the case of PCAN1 and HK215,16 or through interaction with other transcriptional activators such as serum response factor (SRF) or FoxA1 and the androgen receptor (AR)17,18.\n\nTranscriptomic profiling combined with global mapping of > 9,500 genomic binding sites by ChIP-sequencing revealed a set of 282 putative direct target genes that were differentially expressed in young NKX3.1-/- prostates not displaying PIN16,19. A subset of NKX3.1 target genes was also regulated by Myc with both transcription factors showing mutual antagonism16. Since overexpression of Myc cooperates with loss of Nkx3.1 in mouse prostate tumorigenesis, maintaining proper control of the common Nkx3.1/Myc target genes may be involved in Nkx3.1’s tumor suppressor function16. A similar study in aged mice already displaying PIN revealed a gene expression signature indicative of impaired response to oxidative stress20. Interestingly, these changes correlated with a 5-fold increase in oxidative DNA damage in Nkx3.1-/- prostates. Whether oxidative DNA damage is a direct consequence of loss of NKX3.1 or a secondary consequence of PIN development is unknown.\n\nAnother key to understanding the tumor suppressor function of NKX3.1 potentially lies with its protein interaction partners. Several have been described that modulate NKX3.1’s transcriptional effects (e.g. SRF17,21, PDEF14, HDAC19, SP113, MYC16, and AR18). In addition, NKX3.1 was shown to bind to and augment the activity of topoisomerase I, suggesting that it functions in DNA repair22,23. NKX3.1 localizes to sites of DNA damage, promotes ATM and ATR activity, and enhances the survival of cells exposed to DNA damage24. Loss of NKX3.1 function in premalignant prostate cells may therefore accelerate the acquisition of DNA damage, potentially aggravated by unabated accumulation of reactive oxygen species thus promoting cellular transformation24. Nevertheless, it is currently unclear whether the function of NKX3.1 in DNA repair is indirectly mediated through transcriptional effects or directly through physical interactions with the DNA repair machinery.\n\nIn this report, we present an analysis of the NKX3.1 protein interactome that revealed intimate physical links of NKX3.1 with the DNA repair machinery, namely components of the DNA-dependent protein kinase (DNA-PK) holocomplex (XRCC5/Ku80, XRCC6/Ku70) and poly(ADP) ribose polymerase (PARP1). In addition, transcriptomic profiling of immortalized prostate epithelial cells upon acute activation of NKX3.1 revealed a rapid and complex transcriptional response that is a near mirror image of the gene expression signature of human PIN devoid of NKX3.1. Taken together, these data shed new light onto the elusive tumor suppressor activity of NKX3.1, directly implicating this homeoprotein in DNA repair and in driving a gene expression signature indicative of an essential function in maintaining the differentiation state of luminal prostate epithelial cells.\n\n\nMaterials and methods\n\nThe human prostate cancer cell line LNCaP was obtained from ATCC and maintained in RPMI 1640 (Hyclone, Cat.# SH30027.01) supplemented with 10% fetal bovine serum (Sigma, Cat.# F6178-500ML), 50 units/ml penicillin, and 50 units/ml streptomycin (Thermo Scientific HyClone, Cat.# SV30010). The NKX3.1 cDNA was amplified from LNCaP mRNA, sequence confirmed, and cloned into pFLAG thereby attaching three consecutive FLAG epitope tags to the N-terminus. For DNA transfection, LNCaP cells were grown to 50–70% confluence on a 150 mm dish and transfected with 30 µg of plasmid DNA using DOTAP reagent according to the recommendations of the manufacturer (Roche, Indianapolis, IN). Immortalized human prostate epithelial cells (LH cells, kindly provided by Dr. W. Hahn;25) were maintained in Prostate Epithelial Cell Basal Media (Lonza, Cat.# CC-3165) including growth factors, cytokines, and supplements (PREGM Singlequots, Lonza, Cat. # CC-4177).\n\nFor production of adenoviruses, the ADEASY system was used as previously described26. The NKX3.1 cDNA was cloned into the pADTRACK1 shuttle vector. The resulting plasmid was transformed into BJ-ADEASY cells by electroporation. Adenoviral DNA generated by recombination in BJ-ADEASY cells was isolated and transfected into 293 cells (ATCC) using standard calcium phosphate procedures. Virus was harvested from cells and amplified by infection of 293 cells. Amplified virus was tittered and used at a multiplicity of infection of ~100.\n\nThe following antibodies were used: Flag mouse monoclonal (Sigma-Aldrich Cat# F1804, RRID:AB_262044), NKX3.1 mouse monoclonal for immunoblotting (Invitrogen Cat# 35-9700, RRID:AB_138690), Anti-human NKX3.1 goat polyclonal (Santa Cruz Biotechnology, Inc. Cat# sc-15022, RRID:AB_650285) for immunoprecipitation, GFP mouse monoclonal (Clontech Cat# 632380, RRID:AB_10013427), actin mouse monoclonal (MP Biomedicals, Irvine, CA, Cat.# ICN691001), BANF rabbit polyclonal (EMD Millipore Cat# 09-893, RRID:AB_1977041), Ku70 mouse monoclonal (GeneTex Cat# GTX23114, RRID:AB_367103), Ku80 mouse monoclonal (GeneTex Cat# GTX72225, RRID:AB_383445 ), MYC rabbit polyclonal (Epitomics Cat# 1472-1, RRID:AB_562270), p21 rabbit monoclonal (Cell Signaling Technology Cat# 2947S, RRID:AB_823586), HSPA8 rabbit polyclonal (Sigma-Aldrich Cat# SAB2101098, RRID:AB_10604580), PARP mouse monoclonal (BD Biosciences Cat# 556494, RRID:AB_396433), HOXB13 rabbit polyclonal (Invitrogen Cat# 422500, RRID:AB_1500227).\n\nCells of one 150 mm dish transfected with pFLAF-NKX3.1 or empty vector were lysed in each 1 ml IP lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X 100) on ice. Per affinity purification, 4 µg FLAG M2 antibody (Sigma-Aldrich Cat# F1804, RRID:AB_262044) was coupled to 50 μl magnetic beads in 0.2 M triethanolamine, pH 8.2 and 20 mM dimethyl pimelimidate with rotational mixing at room temperature for 30 min. The reaction was stopped by resuspending beads in 1 ml 50 mM Tris, pH 7.5 for 15 min. After five washes in IP lysis buffer, the beads were added to the cell lysate. Upon incubation for 4 h at 4°C, the lysate was removed and stored as “depleted lysates” at -20°C, whereas the beads were washed 5 times with 1 ml IP lysis buffer. After the final wash, beads were resuspended in 50 µl elution buffer (5 µg of triple FLAG peptide in PBS) and incubated at 4°C for 30 minutes with vortexing. The sample was analyzed by immunoblotting (10%), silver staining (2%), and LC-MS/MS (88%).\n\nLC-MS/MS analysis of affinity purified FLAG-NKX3.1 complexes was performed as previously described in detail27,28. In brief, eluates were digested in solution with trypsin, and peptides were separated by reversed phase chromatography. Peptides were analyzed on an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific; San Jose, CA). The MS/MS method was top 4-data dependent. Dynamic exclusion was enabled. Data were searched against an international protein index (IPI) human protein database using Sorcerer-SEQUEST (SageN Research; Milpitas, CA).\n\nSpectral counts are the number of times an ionized peptide is selected by the mass spectrometer for MS/MS, in the data-dependent mode and provide widely accepted, semi-quantitative estimates of relative protein abundance29. QTools, which are in-house developed visual basic macros (available from: www.dieter-wolf-lab.org/protocols) for automated spectral count analysis, were used to compute spectral counts of the proteins, using the PeptideProphet output from the trans-proteomic pipeline (TPP; Institute for Systems Biology, Seattle, WA;30).\n\nPurifications of FLAG-NKX3.1 were performed in quadruplicate (i.e. 4 biological replicates), each time starting with a fresh batch of cells. Altogether eight samples from affinity purifications (quadruplicates of mock and FLAG-NKX3.1) were analyzed repeatedly (3 times per sample, i.e. 3 technical replicates of each sample) by LC-MS/MS for a total of 24 LC-MS/MS runs.\n\nAltogether we identified 315 human proteins (Data set 1A). To compile a high confidence NKX3.1 protein interactome, we first performed a background subtraction, i.e. the spectrum count obtained for each protein in the mock purifications was subtracted from the spectrum count obtained for that same protein in the corresponding FLAG-NKX3.1 purification (Data set 1B). The subtracted spectrum counts were then summed over all 4 independent purifications. If negative values were obtained after summing (i.e. if a protein was consistently more abundant in the mock purification than in the FLAG-NKX3.1 purification), the protein was disregarded. This resulted in a list of 250 proteins with an average spectrum count of 9.94 (Data set 1B). From this lists of background-subtracted data, we removed all proteins with spectrum counts below the average (≤ 10) to exclude low-abundance proteins potentially non-specifically associated with NKX3.1. This resulted in a list of 71 background subtracted and abundance-filtered proteins. In the next step, we collapsed redundant protein database entries (often resulting from multiple protein isoforms that were not distinguished by the peptides identified by LC-MS/MS) into single entries by adding their spectrum counts both in the mock and NKX3.1 purifications. This resulted in a non-redundant list of 58 proteins, which we refer to as the high confidence interactome (Data set 1B).\n\nSince spectrum counts depend on protein size (larger proteins giving rise to more tryptic peptides), we normalized spectrum counts to protein molecular weights, which we have previously found to be an appropriate method of normalization31. The summed, normalized spectrum count numbers of all non redundant proteins were used to assemble the final background subtracted list of 58 NKX3.1 interacting proteins (referred to as Sum NKX3.1 – Mock). The summed normalized spectrum count numbers were also used to determine the fold enrichment of a protein in the NKX3.1 sample over mock (Sum NKX3.1/Mock). Both lists were sorted according to abundance and compared in Figure 1D to illustrate that both methods of background filtering (subtraction or division) yield an overlapping list of high confidence NKX3.1 interactors. The spectrum count intensity map in Figure 1C reiterates most of the steps described above thus presenting a comprehensive view of the analysis.\n\n(A) Representative purification of FLAG-NKX3.1 from transfected LNCaP cells. Cell lysates were absorbed to anti-FLAG M2 resin, and specifically retained proteins were eluted with FLAG peptide and separated by SDS-PAGE. A band migrating with the expected molecular weight of FLAG-NKX3.1 and absent from the mock purification (empty vector) is highlighted. (B) Four-way Venn diagram to indicate the degree of overlap in the protein content detected in four independent purifications of FLAG-NKX3.1. (C) Map of spectrum count intensities in the four independent FLAG-NKX3.1 and mock purifications. The map also contains the sum of spectrum counts across all purifications as well as summed data after adjustment for protein molecular weights. The right most two columns present two distinct ways of background correction, either by subtracting mock values from NKX3.1 values (NKX3.1 – Mock) or by calculating the factor of enrichment in the NKX3.1 sample over mock (NKX3.1/Mock). See the Materials and methods section for details on data analysis and processing. (D) Spectrum count intensity maps of the 25 most abundant components of the NKX3.1 interactome. Data were sorted either by factor of enrichment (left panel, NKX3.1/Mock sorted) or by background subtracted values (right panel, NKX3.1 – Mock sorted). Black type font indicates the proteins occurring on both lists independent of the method of abundance-based sorting.\n\nThe NKX3.1 interactome was analyzed with the Cytoscape Reactome FI plugin32. The list of NKX3.1 interacting proteins was loaded into Cytoscape and used to build Reactome networks allowing linker genes. The networks were clustered into modules, and pathways enriched in the modules (FDR ≤ 0.01) were identified (Figure 2A).\n\n(A) The list of NKX3.1 interacting proteins was loaded into Cytoscape and used to build Reactome Functional Interaction networks. The networks were clustered into modules (indicated by colors), and pathways enriched in the modules (FDR ≤ 0.01) were identified. Diamonds represent network components that were not identified as NKX3.1 interacting proteins. (B) LNCaP cells were transfected with FLAG-NKX3.1 (+) or empty vector (-) followed by absorption of cell lysate to FLAG M2 resin to purify FLAG-NKX3.1. Co-purifying DNA repair proteins were detected by immunoblotting. The bottom four panels are from the same affinity purification resolved on a separate gel. The asterisk denotes an unspecific cross-reactivity of the HSPA8 antibody. Cropped blot images are shown; see Figure S7 for full images. (C) A nuclear protein fraction was prepared from LNCaP cells and employed for immunoprecipitation with NKX3.1 antibodies or an IgG control as indicated. The same samples before (“B”) and after (“A”) immunoprecipitation are shown to document the specific depletion of endogenous NKX3.1. The bottom three panels are from the same immunoprecipitate resolved on a separate gel. Cropped blot images are shown; see Supplement Figure S7 for full images.\n\nDuplicate RNA samples collected from NKX3.1 adenovirus transduced LH cells or from LH cells transduced with the GFP control virus were used for microarray analysis on the Illumina platform. The Human 6-V2 Expression BeadChips (Illumina) were used, which contain ~46,000 transcript probes. Primary data was collected using the manufacturer’s BeadArray Reader using the supplied scanner software. Data analysis was done in three stages. First, expression intensities were calculated for each transcript probed on the array for all hybridizations using Illumina’s Beadstudio#2 software. Secondly, intensity values were quality controlled and normalized. Quality control was carried out by using the Illumina Beadstudio detection p-value set to < 0.05 as a cutoff. This removed probes whose signals were too low to be reliably detected on the array. After this step, the initial ~46,000 probes were reduced to 22,319 (Data set 2A). Measurements were then normalized using the normalize.quantiles routine from the Affymetrix package in Bioconductor. This procedure accounted for any variation in hybridization intensity between the individual arrays. An assessment of several different normalization techniques using the Bioconductor maCorrPlot routine suggested that normalize.quantiles was the most appropriate for the data. Finally, these normalized data were imported into GeneSpring and analyzed for differentially expressed genes. The raw datasets were submitted to the GEO database (accession number GSE47030).\n\nTo identify genes differentially expressed between LH cells infected with Ad-GFP and Ad-GFP-NKX3.1 the biological replicates for each time point (7 h and 10 h) were averaged. Datasets were interrogated for genes with statistically significant differences between the two groups (i.e. +/- NKX3.1) based on the results of the Welch t-test (parametric test, variances not assumed equal; p-value cutoff 0.05). To find the genes with the most robust changes in expression, the data was plotted as a “Volcano Plot” (Supplementary Figure S2B), which allows statistical significance to be measured along with the extent of fold change in expression. Lists of mRNAs significantly changing 3-fold or 5-fold upon expression of NKX3.1 were assembled (Data set 2C).\n\nLH cells were infected with 20 µl of Ad-GFP or Ad-GFP-NKX3.1 viruses and total RNA was isolated after 6, 8, 10, and 12 h using the RNeasy mini kit (Qiagen, Valencia, CA). RNA concentrations were determined by measuring absorption at 260 nm in a spectrophotometer. Aliquots of 2 μg of total RNA from each sample were reverse-transcribed into cDNA using an Omniscript RT kit (Qiagen) according to the manufacturer's instructions. Quantitative Real-Time PCR was performed using Brilliant SYBR Green QPCR Master Mix (Stratagene, La Jolla, CA) and the Mx3000 Real-Time PCR System (Stratagene). Gene specific primers were designed using the Primer3 algorithm (http://frodo.wi.mit.edu/) as shown below. PCR reactions were run according to the protocol for the Brilliant SYBR Green QPCR Master Mix. Briefly, PCR was carried out using a final concentration of 0.2 μmol of the primer pairs, 50 ng of cDNA template and 12.5 μl of Brilliant® SYBR Green QPCR Master Mix. The volume was adjusted to 25 μl by adding RNase-free water. The thermocycling protocol began with a 3 min denaturation at 95°C, a 40 cycle amplification program consisting of 30 s denaturation at 95°C, 1 min annealing at 55°C and 30 s extension at 95°C. Upon conversion of raw ct values to linearly related X(0) values, expression values were normalized to GAPDH, and expression changes were expressed as ratios of mRNA levels in NKX3.1 infected versus GFP infected cells (NKX3.1/GFP). The ratios were log2 transformed and averaged across two technical replicates, and standard deviations were calculated.\n\nPrimer sequences used for Q-PCR:\n\nHSPA6_F CCGTGAAGCACGCAGTGAT\n\nHSPA6_R ACGAGCCGGTTGTCGAAGT\n\nTAGLN_F GCTGGAGGAGCGACTAGTGG\n\nTAGLN_R CCTCCTGCAGTTGGCTG\n\nCDH2_F TGGAACGCAGTGTACAGAATCAG\n\nCDH2_R TTGACTGAGGCGGGTGCTGAATT\n\nCCND2_F TACCTTCCGCAGTGCTCCTA\n\nCCND2_R TCACAGACCTCCAGCATCCA\n\nSTAT2_F CACCAGCTTTACTCGCACAG\n\nSTAT2_R TGGAAGAATAGCATGGTAGCCT\n\nEEF1A2_F GCTGAAGGAGAAGATTGACC\n\nEEF1A2_R TTCTCCACGTTCTTGATGAC\n\nCDKN1A_F TTGTCTTTCCTGGCACTAAC\n\nCDKN1A_R CCCTCGAGAGGTTTACAGTC\n\nHES1_F GCATCTGAGCACAGAAAGTC\n\nHES1_R CTGTCATTTCCAGAATGTCC\n\nS100A2_F GGGAAATGAAGGAACTTCTG\n\nS100A2_R CACATGACAGTGATGAGTGC\n\nTNFa_F1 GTGGACCTTAGGCCTTCCTC\n\nTNFa_R1 ATACCCCGGTCTCCCAAATA\n\nTNFa_F2 CCCAGGCAGTCAGATCATCTT\n\nTNFa_R2 TCTCAGCTCCACGCCATT\n\nLH cells were seeded in 384-well plates at a density of 2000 cells per well. After 24 hours, cells were transduced with Ad-GFP-NKX3.1 or control Ad-GFP adenoviruses for the times indicated in Figure 6D–F. Proliferation (i.e. DNA synthesis) was measured using the Click-iT® EdU Alexa Fluor® 594 HCS kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Briefly, 10 µM 5-ethynyl-2′-deoxyuridine (EdU) was added to culture media for one hour, and cells were fixed with 3.7% formaldehyde, washed with PBS twice, permeabilized with 0.1% Triton X-100 in PBS, stained with Click-iT Alexa Fluor 594 dye, and counterstained with 1 µg/mL Hoechst 33342 (Blue). Plates were scanned and analyzed by using a Celigo automated cytometer at dual wave length to detect Hoechst dye (total cell count) and Alexa Fluor 594 (cells incorporating EdU and thus undergoing DNA synthesis). Four images per well were obtained at each wave length, and the percentage of proliferating cells was calculated by dividing the number of Alexa positive cells by the total cell number.\n\nMAP kinase inhibitors and neutralizing antibodies were added two hours after viral transduction. JNK inhibitors SP600125 (EMD Chemicals Inc, San Diego, CA) and p38 inhibitor SB203580 (Enzo Life Sciences, Farmingdale, NY) were used at 20 µM. Mouse IgG directed against TNFα (Clone 6401, R&D Systems, Minneapolis, MN) and whole mouse IgG as a control (Jackson ImmunoResearch Laboratories, West Grove, PA) were used at 5 µg/ml.\n\nIngenuity Pathway Analysis (IPA, Ingenuity Systems) was used for pathway and network analysis. The bulk of the analysis was performed with the 5× dataset (mRNAs showing a significant ≥ 5-fold change upon expression of NKX3.1). The 3× dataset was used for the MYC network. Datasets were imported into IPA, and analyzed with the following settings: Reference Set: Ingenuity Knowledge Base (Genes + Endogenous Chemicals); Network Analysis: Direct and Indirect Relationships; Data Source: Ingenuity Expert Findings; Confidence: Experimentally Observed; Species: Mammal (human, mouse, rat) and Uncategorized (e.g. chemicals); Tissue and Cell Lines: All.\n\nThe 5× dataset was uploaded to the NextBio server through the Sanford-Burnham portal. 153 of the 158 features of the 5× dataset were recognized and could be interpreted by NextBio. The analysis was performed using default settings. Significantly enriched transcription factor binding sites were identified through corresponding Biogroups. The overlap between the 5× dataset and the gene expression study by Nanni et al.33 was identified through a search against all curated studies.\n\nFlag-NKX3.1 transfected LNCaP cells were seeded onto 15 mm poly-lysine coated glass cover slips, and fixed using formaldehyde (3.7% in PBS). Samples were stained with mouse monoclonal FLAG (Sigma) or goat polyclonal NKX3.1 antibodies (Santa Cruz). Alexa Fluor 568 (red) donkey anti-mouse IgG and Alexa Fluor 488 (green) donkey anti-goat IgG conjugate antibodies (Life Technologies Cat# A10037, RRID:AB_11180865 and Cat# A11055, RRID:AB_10564074) were used as secondary antibodies. The nuclei were stained with 4’–6’ diamidino-2-phenylindole (DAPI). Samples were imaged on a Nikon Type 120 inverted fluorescent microscope using 60× magnification.\n\n\nResults\n\nReasoning that the NKX3.1 interactome may be most effectively profiled in cells that naturally express this protein, we transiently expressed FLAG epitope-tagged NKX3.1 in LNCaP human prostate cancer cells. FLAG-NKX3.1 was approximately 5-fold in excess over endogenous NKX3.1 (Supplementary Figure S1A) but localized primarily to cell nuclei (Supplementary Figure S1B). The proteasome inhibitor MG132 was added 4 hours prior to lysate preparation in order to slow the rapid clearance via the ubiquitin-proteasome pathway to which NKX3.1 is normally subjected34,35. Cell lysate was absorbed to anti-FLAG M2 resin, and specifically retained proteins were eluted with FLAG peptide. Four independent affinity purifications were performed in parallel with mock purifications of lysate of cells transfected with empty vector. The eluates were examined by SDS-PAGE (Figure 1A) and subjected to LC-MS/MS analysis in order to determine their protein composition. Altogether, 315 proteins were identified at a false-positive rate of ≤ 0.01 (Data set 1A).\n\nThe protein dataset was subjected to background subtraction and abundance-based filtering to arrive at a list of 58 high confidence NKX3.1 interacting proteins (see Materials and methods and Data set 1B). Fifty five of the 58 proteins were identified in at least two independent purifications, and 27 were identified in at least three purifications (Figure 1B, Data set 1C). Five proteins were consistently identified as NKX3.1 interaction partners in all four independent purifications, namely NKX3.1, the DNA repair proteins XRCC5/Ku80 and PARP1, and the protein synthesis proteins RPS9 and PABPC1.\n\nWe next performed a relative quantification of the NKX3.1 interactome based on spectral counting29. Upon summing the molecular weight adjusted spectrum counts of each protein across the four mock and NKX3.1 purifications, we derived background corrected quantifications by either subtracting summed mock values from summed NKX3.1 bait values (NKX3.1 – Mock) or by dividing NKX3.1 bait values from mock values (NKX3.1/Mock) to obtain the factor by which a protein was enriched in the NKX3.1 bait samples over the mock sample. Both methods confirmed the expectation that NKX3.1 was the most abundant protein identified in the FLAG affinity purifications (Figure 1C, D). We also performed Reactome Functional Interaction analysis to construct a functional interaction network of NKX3.1 binding proteins derived from manually curated literature data32. The network was clustered into modules and enriched functional pathways/reactions were identified (Figure 2A).\n\nAmong the 10 most abundant co-purifying proteins were the components of the DNA-dependent protein kinase (DNA-PK) holoenzyme, XRCC5/Ku80, XRCC6/Ku70, and poly(ADP) ribose polymerase (PARP1) (Figure 2A). DNA-PK and PARP1 have important functions in DNA double strand break repair, recombination, and telomere maintenance but are also involved in chromatin and transcriptional control36–38. For example, Ku proteins associate with a series of homeodomain proteins (HOXC4, OCT1, OCT2, DLX2) thereby recruiting them to DNA ends where they are phosphorylated by DNA-PK39. Such phosphorylation was proposed to lead to DNA damage-dependent changes in their transcriptional activities. ADP-ribosylation mediated by PARP1 can stimulate the ability of DNA-PK to phosphorylate protein substrates40. Our interactome data provide a possible mechanism underlying the previously observed localization of NKX3.1 to sites of DNA damage24, although the functional consequences of these interactions for NKX3.1 transcriptional activity remain to be established. Regardless, follow-up co-immunoprecipitation experiments showed that overexpressed NKX3.1 readily interacted with endogenous XRCC5/Ku80, XRCC6/Ku70, and PARP1 (Figure 2B). Interaction of DNA-PK with ectopically expressed NKX3.1 was very recently reported in an independent study41. We show here that endogenous NKX3.1 also interacts with XRCC5/Ku80, XRCC6/Ku70, and PARP1 (Figure 2C).\n\nAmong the top ranking NKX3.1 interacting proteins was also interleukin enhancer binding factor 2 (ILF2/NFAT 45 kDa) (Figure 1D). This protein was previously shown to interact with the DNA-PK-Ku complex42 and to be part of a ribonucleoprotein assembly containing heterogeneous nuclear ribonucleoproteins (hnRNPs), the heat shock protein HSPA8, the poly-A binding protein PABC1, nucleolin (NCL), and several ribosomal proteins43, all of which were also identified here as components of the NKX3.1 interactome (Figure 1C,D, Data set 1A). Most of these interactions were also represented in the Reactome network (Figure 2A). Two additional subunits of this particle, ILF3 and YBX1 were also identified, albeit at low levels (Data set 1A). hnRNPs function in multiple processes, including mRNA splicing, dynamics, stability, and translation, telomere maintenance, DNA repair, and chromatin remodeling and transcription44. They are also major constituents of the nucleolar proteome, which additionally comprises many of the NKX3.1 interacting proteins listed above, including the DNA-PK complex, PARP1, HSPA8, and ribosomal proteins as well as the RNA helicases DDX3 and DDX545,46. Although the significance of these interactions remains unclear, they may reflect a close physical coupling of NKX3.1-dependent mRNA transcription to mRNA processing47 and/or hitherto unappreciated role for NKX3.1 in nucleolar ribosome biogenesis and cytoplasmic mRNA transport. A similar proposition was made to rationalize the interactome of the transcription factor SOX2, which shares remarkable overlap with the NKX3.1 interactome48.\n\nAnother highly abundant NKX3.1 interactor is the chromatin and nuclear assembly regulator BANF1 (Figure 1D). This interaction was confirmed by co-immunoprecipitation (Figure 2B). BANF1 was previously shown to bind two other proteins identified in the NKX3.1 interactome, emerin (EMD) and thymopoetin (TMPO)49. In addition, BANF1 interacts with several other homeodomain transcription factors and regulates the transcriptional activity of one of them, CRX50. It is thus likely that BANF1, in complex with emerin and thymopoetin, is involved in NKX3.1-mediated gene regulation. The nuclear matrix attachment proteins SAFA/HNRNPU and SAFB, which were also identified as NKX3.1 interacting proteins, may also participate in this process.\n\nFinally, we identified an interaction of NKX3.1 with the homeobox transcription factor HOXB13 (Data set 1C). This interaction was confirmed by co-immunoprecipitation (Figure 2A). HOXB13 also interacts with the androgen receptor and regulates the cellular response to androgen51. In addition, germline mutations of HOXB13 significantly increase risk of hereditary prostate cancer through unknown mechanisms52. However, further studies discounted the intriguing possibility that mutation of HOXB13 alters its interaction with NKX3.1 (CCY & DAW, unpublished observation).\n\nPrevious determinations of NKX3.1-dependent gene expression signatures have profiled prostates of mice that developed and aged in the complete absence of NKX3.116,19,20. These signatures may therefore describe adaptive changes that occur in response to long-term depletion of NKX3.1 in addition to its immediate effects on gene expression. We have therefore chosen to acutely introduce NKX3.1 into immortalized human prostate epithelial cells (LH cells25) that do not express detectable levels of NKX3.1 protein (data not shown). We produced adenoviruses driving the expression of either GFP alone or GFP and NKX3.1 from separate promoters (Ad-GFP and Ad-GFP-NKX3.1 viruses, respectively). LH cells were infected with these viruses according to the scheme in Figure 3A. GFP signal became first detectable by live cell fluorescence microscopy 6 hours after infection (data not shown). We therefore harvested duplicate cultures of cells for immunoblotting 7 and 10 hours after infection and determined that NKX3.1 and GFP were expressed at both time points (Figure 3B). No cytopathic effects of adenovirus infection were observed within the time frame of the experiment. In parallel, we prepared duplicate RNA samples of the 7 hours and 10 hours time points for transcriptome analysis.\n\n(A) Schematic representation of the time course of the experiment. LH cells were infected in duplicate with adenoviruses driving the expression of either GFP alone or GFP and NKX3.1 from two separate promoters. GFP expression became first apparent by fluorescence microscopy 6 hours after transfection (data not shown). (B) Duplicate cell lysates were prepared 7 and 10 hours after infection, and examined for the expression of GFP and NKX3.1 by immunoblotting. NKX3.1 expression was already detectable at the earliest time point (7 hours). (C) Quantitative RT-PCR analysis of 9 mRNAs whose expression is changed in response to NKX3.1. LH cells were infected with adenoviruses driving the expression of either GFP alone or GFP and NKX3.1, and mRNA was isolated after the indicated time points (6, 8, 10, 12 hours). The RNA samples were analyzed by Q-PCR, and expression values are shown as log2 transformed ratios of the mRNA level in NKX3.1 infected versus GFP infected cells (NKX3.1/GFP). Error bars indicate standard deviations obtained from two replicate measurements. The left panel shows data for 5 mRNAs that were upregulated by NKX3.1 in the array dataset, whereas the right panel shows data for four mRNAs that were downregulated.\n\nThe global changes in transcript levels noted in response to NKX3.1 expression were very similar at the 7 hours or 10 hours time points (Supplementary Figure S2A). Statistically significant changes were observed for several hundred mRNAs. To reduce the number of mRNA changes to be further interrogated to a manageable number, we arbitrarily set a cut-off of 5-fold change. This yielded lists of 158 differentially expressed genes for the 7 hours time point (Supplementary Figure S2B) and 165 for the 10 hours time point. Since there was a considerable overlap of both lists, we limited the further analysis to the 7 hours sample. Data sets 2A and B summarize all mRNA expression data. Supplementary Table 1 presents a ranked list of all 107 mRNAs with > 5-fold upregulation, whereas Supplementary Table 2 presents a corresponding list of all 51 mRNAs with > 5-fold downregulation in NKX3.1 expressing LH cells (see also Data set 2C). We chose 5 upregulated and 5 downregulated mRNAs for validation by Q-PCR with a fresh set of replicate RNA samples prepared from cells infected with Ad-GFP or Ad-GFP-NKX3.1 for increasing periods of time. Nine out of the 10 expression changes confirmed the tendency seen from microarrays, although variability was substantial for some measurements (Figure 3C). We failed to confirm the induction of KRT17 mRNA apparent from the array data (not shown). Additional validation by Q-PCR and immunoblotting is shown in various sections below (see Figure 6).\n\nExamination of the lists of mRNA changes revealed a fundamental reprogramming of gene expression in LH cells upon acute expression of NKX3.1. Overall, the changes were indicative of inhibition of cell proliferation and induction of cell differentiation. For example, 9 epithelial differentiation markers (cytokeratins 5, 6B, 7, 8, 17, 18, 19, stratifin, kallikrein 5) were strongly induced. In addition, the Notch pathway, which is often downregulated in prostate cancers53, was induced (DLL1, HES1, JAG2). The cyclin-dependent kinase inhibitor p21 (CDKN1A), which inhibits cell cycle progression and induces cell differentiation54, was also increased.\n\nReassuringly, many of the strongest NKX3.1-induced mRNAs encode proteins that were previously shown to be downregulated in human prostate cancer based on immunohistochemistry (Supplementary Table 1). This included, for example, the calcium binding proteins S100A2 and A1455, the 14-3-3 protein stratifin56,57, laminin A58, claudin 759, prostasin60, P cadherin61, and kallikrein 562. Cyclin D2 is considered an activator of cell cycle progression but was induced by NKX3.1. Remarkably, however, cyclin D2 is typically downregulated in human prostate cancers63. Four mRNAs encoding HSP70s were upregulated (Supplementary Table 1). HSP70 expression is frequently lost in aggressive prostate cancers64 and experimental HSP70 overexpression inhibits the tumorigenicity of prostate cancer xenografts in mice65. Likewise, three genes encoding the HSP70 co-chaperones DnaJ/HSP40 were upregulated > 5-fold. Lastly, two glutathione transferases were upregulated by NKX3.1, a finding that is consistent with the previous demonstration that NKX3.1 upregulates oxidative stress defense20.\n\nThe list of downregulated genes (Supplementary Table 2) included genes involved in cell migration (actin/myosin-related, collagens 1A1, 5A1, 5A2), several growth factors, and the interferon/STAT pathway. Many of the most downregulated genes were previously shown to be overexpressed in prostate and other cancers (Supplementary Table 2). This applies, for example, to eukaryotic translation elongation factor 1 alpha (EEF1A2) which is a potential oncogene66, the BMP antagonist gremlin 167, and the transcription factor FOXD168. N-cadherin, which is frequently found to replace epithelial cadherin forms in prostate cancers (“cadherin switch”) was also strongly downregulated69. Significantly, NKX3.1 also upregulated P cadherin thus reversing the cadherin switch.\n\nWe also compared our list of 331 mRNAs that were changed ≥ 3-fold by NKX3.1 with a recent list of 282 mouse genes thought to be direct NKX3.1 targets based on a combination of expression and ChIP-seq data16. Despite the species difference and the diametrical strategies (overexpression versus knockout), 10 genes were represented on both lists (Supplementary Table 3). This overlap is highly significant when considering that 8 out of these 10 genes were regulated by NKX3.1 in the same direction.\n\nTo assess functional modules and signaling pathways affected by NKX3.1, we performed a global analysis with the Ingenuity Pathway Analysis (IPA) package. The analysis was performed with the dataset of mRNAs changing more than 5-fold (“5× dataset”) or, where indicated, with a larger dataset of mRNAs changing more than 3-fold (“3× dataset”, 357 genes). Since identical top scoring pathways were obtained with both datasets, the analysis was largely restricted to the smaller 5× dataset, unless otherwise noted.\n\nConsistent with the involvement of NKX3.1 in prostate development, we found highly significant overrepresentation of IPA “Functions” pertaining to development, cell movement, proliferation and cell growth (Figure 4A). Of particular interest was the term “Reproductive Systems Disease”, which included the subgroup “Prostatic intraepithelial neoplasia” (PIN). PIN is the earliest known precursor lesion of prostate cancer, and frequently shows decreased NKX3.1 levels70. The “PIN” Function contained the seven genes listed in Figure 4B. A previous study determined that six of these genes were downregulated in PIN versus normal prostate, whereas one was upregulated71. Remarkably, five out of the seven genes displayed a mirror image of the changes occurring in PIN when examined in NKX3.1-expressing LH cells (Figure 4B). These findings suggest that changes in gene expression in early PIN may be causally linked to loss of NKX3.1.\n\n(A) Select IPA “Functions” significantly overrepresented in the 5× mRNA set. (B) List of mRNAs with inverse expression in prostatic intraepithelial neoplasia (PIN;71) and NKX3.1 expressing LH cells. mRNAs shown in red are upregulated whereas those shown in green are downregulated. (C) Select IPA “Canonical Pathways” overrepresented in the 5× dataset. The abscissa on the top indicates the percent fraction of all possible pathway components that were represented in the dataset. Since this dataset only contained a relatively small number of 158 mRNAs, a small percent wise overrepresentation of pathway components is statistically highly significant (p < 0.05, see yellow graph).\n\nAs shown in Figure 4C, a number of pathways were overrepresented that were not readily apparent from the manual curation of the gene lists presented above. For example, the analysis indicated upregulation by NKX3.1 of the p53 and IL1 pathways, in addition to the Notch signaling pathway. Interferon signaling, in turn, appeared to be switched off by acute NKX3.1 expression.\n\nTNFα network. To obtain a better understanding of the regulatory circuitry underlying NKX3.1-induced modulation of particular functional pathways, we performed network analysis using Ingenuity IPA software. The highest ranking network presented in Figure 5A featured TNFα, a gene that was induced by NKX3.1 (Supplementary Table 1, Figure 6A), in the center with edges reaching to 27 distinct nodes. Eighteen of these edges were defined by a gene regulatory relationship (i.e. expression edge) thus signifying genes that are known to be either induced or suppressed by TNFα signaling. Further annotation of the TNFα network also connected TNFα to NKX3.1-induced suppression of cell movement through downregulation of action-myosin based mobility components and enhancement of cell adhesion through upregulation of laminins (Figure 5A). Both processes are considered bona fide hallmarks of tumor suppression. Close examination of every TNFα expression edge revealed considerable concordance between the definition of the edge (based on the published literature) and the actual expression of the target node in response to NKX3.1. Fourteen first degree nodes predicted to be activated by TNFα were also upregulated by NKX3.1 (Supplementary Table 4). Consistent with MAP kinase signaling being a major downstream pathway activated by TNFα, we found that a chemical inhibitor of JNK but not p38 could partially antagonize NKX3.1-induced expression of HSPA6 and HES1 (Figure 6B).\n\n(A) TNFα network. Node colors represent the level of up- (red) or down- (green) regulation upon expression of NKX3.1. (B) Tumor suppressor p53 network. The p53-TERT-EGF-JUN quadrangle is highlighted by dark blue edges. (C) MYC network. First degree edges of MYC are highlighted in light blue. (D) PDGFB/TGFβ network. First degree edges are highlighted in light blue, the PDFGB-TGFβ link in dark blue.\n\n(A) Quantitative RT-PCR analysis of TNFα mRNA. LH cells were infected with adenoviruses driving the expression of either GFP alone or GFP and NKX3.1, and mRNA was isolated after the indicated time points (6, 8, 10, 12 hours). The RNA samples were analyzed by Q-PCR with two different primer sets amplifying TNFα mRNA, and expression values are shown as log2 transformed ratios of the mRNA level in NKX3.1 infected versus GFP infected cells (NKX3.1/GFP). Error bars indicate standard deviations obtained from two replicate measurements. (B) LH cells were infected with adenoviruses driving the expression of either GFP alone or GFP and NKX3.1. After 4 hours, 10 μM of the JNK inhibitor SP600125 or 10 μM of the p38 kinase inhibitor SB203580 were added followed by mRNA isolation after 6 hours. The levels of HSPA6 and HES1 were analyzed by Q-PCR. Expression values are shown as log2 transformed ratios of the mRNA level in NKX3.1 infected versus GFP infected cells (NKX3.1/GFP). Error bars indicate standard deviations obtained from two replicate measurements. (C) LH cells were infected with adenoviruses driving the expression of either GFP alone or GFP and NKX3.1, and protein lysates were prepared after the indicated time points (6, 8, 10, 12 hours). The expression of the indicated proteins was determined by immunoblotting. Cropped blot images are shown; see Figure S8. for full images. (D) LH cells were infected with Ad-GFP and Ad-GFP-NKX3.1 viruses, and the rate of DNA synthesis was measured by EdU incorporation after the indicated times (top graphs). The percentage of GFP positive cells was determined as a measure of infection efficiency (bottom graphs). (E) LH cells were infected with Ad-GFP-NKX3.1 virus, and the effect of JNK inhibitor (SP600125, 20 μM) or p38 kinase inhibitor (SB203580, 20 μM) on NKX3.1-mediated suppression of DNA synthesis was measured by EdU incorporation. The percentage of GFP positive cells was determined as a measure of infection efficiency (bottom graphs). (F) LH cells were infected with Ad-GFP-NKX3.1 virus, and the effect of neutralizing antibodies to TNFα or control IgG on NKX3.1-mediated suppression of DNA synthesis was measured by EdU incorporation. The percentage of GFP positive cells was determined as a measure of infection efficiency (bottom graphs).\n\np53 network. Another high scoring network featured the tumor suppressor p53 at the center with first degree edges to 8 nodes. Although p53 was upregulated neither at the mRNA nor protein level (Figure 6C), a finding which is consistent with the well-established activation of p53 at the post-translational level, the network indicated robust induction of some of its known target genes. As shown in Figure 5B, this included the 14-3-3 sigma protein stratifin (SFN), an epithelial differentiation marker missing from many prostate cancers56,72, the cyclin-dependent kinase inhibitor p21 (CDKN1A,73), and the p53 apoptosis effecter PERP74. Induction of p21 protein by NKX3.1 was confirmed by immunoblotting (Figure 6C). Annexin A8 (ANXA8) is also known to be upregulated by p5375. Using the 3× dataset, we pinpointed an additional 7 mRNAs that are upregulated by NKX3.1 as known targets of p53 (Supplementary Figure S3). These findings suggested that the p53 tumor suppressor pathway is activated by acute induction of NKX3.1 in LH cells. The network contained three additional highly connected nodes, telomerase (TERT), EGF, and JUN, which formed a quadrangle with p53. Although JUN mRNA was not induced by NKX3.1, a positive effect of p53 on JUN was reported previously76.\n\nMYC network. A further high scoring network that was obtained with the 3× dataset was organized around the MYC oncogene (Figure 5C). MYC itself was 4-fold downregulated by NKX3.1 expression, an effect that was validated by immunoblotting (Figure 6C). This coincided with downregulation of several genes that were previously found to require MYC function for their expression (TXNIP, IFI1677). In addition, the MYC interaction partner PARP10 was downregulated upon expression of NKX3.1. Conversely, two genes that are negatively regulated by MYC were activated upon NKX3.1 expression (PERP78, NDRG79), suggesting that NKX3.1-induced downregulation of MYC relieves its repressive effect on these genes. In aggregate, these findings suggest that restoration of NKX3.1 expression in LH cells led to downregulation of pathways normally turned on by MYC. This may contribute to a block in proliferation and promote cell differentiation by NKX3.1. Antagonism of NKX3.1 and MYC in target gene regulation and prostate tumorigenesis was recently also demonstrated in a mouse model16.\n\nPDGFB/TGFβ network. Another network featured PDGFβ (PDGFB and PDFGBB), which was induced 5.1-fold by NKX3.1. The induction of PDGFB mRNA and the expression of many of its first degree interacting nodes, is consistent with PDGFB signaling being upregulated by NKX3.1. For example, three nodes that were upregulated by NKX3.1 (CRYAB, SERPINA3, CDKN1A) and two nodes that were downregulated (DAB2, TAGLN) were previously shown to be controlled by PDGFB in the same manner (Supplementary Figure S4;80,81). PDGFB is also known to activate PPAR/RXRα-dependent transcription. Notably, RXRα is itself upregulated by NKX3.1 (5.7-fold), hence explaining the overrepresentation of PPAR signaling in the canonical pathway analysis above (Figure 4C). Since PPAR signaling is known to suppress prostate cancer cell proliferation82, it may be relevant to NKX3.1-mediated tumor suppression.\n\nPDGFB shares a number of nodes with another growth factor, TGFβ (Figure 5D). Although TGFβ1 mRNA was not altered by NKX3.1, the more abundantly expressed TGFβ2 was downregulated (Supplementary Table 5). Most first-degree nodes emanating from TGFβ were downregulated by NKX3.1 expression (Supplementary Figure 3). An additional 25 genes in the TGFβ signaling pathway were either downregulated or unchanged by NKX3.1, further suggesting that NKX3.1 does not activate TGFβ signaling (Supplementary Table 5). Since TGFβ is a strong driver of the epithelial-to-mesenchymal transition (EMT,83), NKX3.1-mediated suppression of TGFβ signaling may contribute to its differentiation-inducing activity.\n\nIn an attempt to obtain a more cohesive view of the global effects of NKX3.1 on prostate gene expression, we merged individual networks. For simplicity, only expression edges were included in Figure 7A. Not only were TNFα and p53 directly linked through an expression-based edge, but several of their individual first degree nodes were targets of edges emanating from both TNFα and p53. For example, TFP12 and CASP4 are positively regulated by both TNFα and by p5376,84–86.\n\n(A) The merged TNFα-p53 network. Network links are highlighted in yellow. Direct edges between TNFα, p53, and JUN are emphasized in blue color. (B) Construction of a network containing the major factors implicated in the NKX3.1 transcriptional program, including FOS/AP1, MYC, and p53. Modules activated by NKX3.1 expression are shaded in red and those suppressed in green. (C) Tentative framework of NKX3.1-dependent changes to cellular modules. Based on the induction of TNFα and FOS mRNA by NKX3.1, and the antagonistic effects of JNK inhibitors on NKX3.1-mediated gene expression and cell proliferation, the framework proposes that TNFα signaling results in activation of AP1 and modulation of downstream genes and functional modules (red squares symbolize upregulation/activation, green squares downregulation). Additional pathways (stippled lines) may impinge on SRF and other transcription factors (not shown).\n\nThe AP1 transcription factor subunit JUN, which was part of the p53 network (Figure 5B) was linked to TNFα resulting in a triangular configuration (Figure 7A). Whereas both TNFα and p53 are known to stimulate the expression of JUN and AP1 activity76,87, NKX3.1 expression did not significantly affect the mRNA level of cJUN (-1.21-fold change) or JUND (+1.25-fold change). However, the JUN interaction partner FOS was increased 3.9-fold by NKX3.1. Since FOS maintains exactly the same edges within the network as JUN (data not shown), AP1 transcriptional activity appears to be upregulated in response to NKX3.1 expression.\n\nFinally, we manually integrated the TNFα network with the connections to all major factors the network analysis had implicated in the NKX3.1 transcriptional program, including FOS/AP1, MYC, and p53. Despite the complexity of the resulting network, a tentative framework for NKX3.1-induced transcriptomic changes is becoming readily apparent (Figure 7B, C). According to this framework, NKX3.1 expression in LH cells results in the activation of the TNFα pathway. This in turn leads to activation of the p53, Notch, PDGFB, and AP1 pathways. Conversely, the MYC and interferon/STAT pathways are turned off. Through Q-PCR and immunoblotting, we have already confirmed several of these predictions (see Figure 3C for p53, Notch, PDGFB, STAT, and Figure 6 for TNFα, MYC, and p53). In addition, transduction with NKX3.1 expressing virus led to growth inhibition of LH cells relative to virus expressing GFP alone (Figure 6D). Notably, growth inhibition was partially rescued by JNK inhibitor and by a neutralizing antibody against TNFα (Figure 6E, F). These observations further support a role of NKX3.1 in inducing a block to cell division and promoting cell differentiation via a TNFα/JNK/AP1-dependent pathways.\n\nWe next employed the NextBio platform to relate our expression data to previously published large-scale genomics data. One dataset that matched with high statistical significance (p = 4.5E-11) featured a set of 1082 genes containing evolutionarily conserved genomic binding sites for AP188. Twenty six of these genes were represented in our list of ~150 NKX3.1 responsive genes with 20 being induced by NKX3.1 (Supplementary Table 1, Supplementary Table 2, Supplementary Figure 5A, Data set 2D). Combined with the evidence from network analysis and the upregulation of FOS, these findings suggest that NKX3.1 causes AP1 activation and/or cooperates with AP1 in gene activation. Consistent with this conjecture is the well-known induction of JUN N-terminal kinase (JNK) activity by TNFα signaling, which enhances the transcriptional activity of JUN. Finally, NFκB which is also induced by TNFα signaling, can cooperate with AP1 at some promoters89.\n\nA second DNA binding motif that was overrepresented (p = 1.6E-5) in NKX3.1 responsive genes conforms to a binding site for serum response factor (SRF). 216 human genes contain the serum response element (SRE) motif in a promoter proximal context that is conserved in mouse, rat, and dog88. These 216 genes included 9 genes that were represented on our dataset, all but one of which was suppressed by NKX3.1 (Supplementary Table 2, Supplementary Figure 5B, Data set 2E). Since NKX3.1 is known to physically interact with SRF17, our data strongly suggests that NKX3.1 cooperates with SRF in transcriptional suppression.\n\nNextbio analysis also revealed a highly significant match with a study comparing gene expression in human prostate cancer tissues33. This study profiled 22 cell lines derived from surgical samples of prostate cancer patients with clinically localized disease and absence of hormonal neo-adjuvant treatment before surgery. In keeping with these selection criteria for early cancers, the cell lines (and primary tumors they were derived from) had suffered loss of 8p21 (i.e. NKX3.1) but did not display genetic abnormalities typical of more advanced prostate cancers (e.g. loss of PTEN, amplification of MYC and androgen receptor). 3415 mRNAs were significantly changed in prostate cancer cell lines relative to normal prostate.\n\nOf 153 differentially expressed genes in our dataset, 82 (53%) were also changed in prostate cancer derived cell lines (PCaDCL), a highly significant overlap (p = 2.0E-36, Supplementary Figure 6; Data set 2F). Of the 82 overlapping genes, 60 were downregulated and 22 were upregulated in PCaDCL versus PrEC. Strikingly, 93% of the mRNAs downregulated in PCaDCL were induced by expression of NKX3.1 in LH cells (Supplementary Table 1). In addition, 19 of the 20 genes upregulated in PCaDCL were downregulated by NKX3.1 (Supplementary Table 2). Moreover, many of the mRNA expression changes observed in the PCaDCL microarray study were independently confirmed at the protein level by immunohistochemistry of prostate cancer tissue samples (Supplementary Table 1 and Supplementary Table 2). These analyses strongly suggest that the principal gene regulatory networks that are affected by NKX3.1 expression in LH cells are inversely perturbed in early human prostate cancer marked by loss of this tumor suppressor.\n\n\n\n\nDiscussion\n\nWe have employed a series of global approaches to explore the tumor suppressor function of NKX3.1. The NKX3.1 interactome revealed a complex pattern of interactions with DNA repair proteins and with other transcriptional regulators such as ILF2 and BANF1 that predict a similarly complex transcriptional program enacted by NKX3.1. Indeed, global analysis of the gene expression pattern actuated by acute expression of NKX3.1 in immortalized human prostate epithelial cells with a basal phenotype (LH cells25,90) revealed a rapid and extensive re-programming with 158 mRNAs changing ≥ 5-fold and 331 mRNAs changing ≥ 3-fold. This complex pattern was interrogated by network analysis to account for the recognition that representation of cellular processes and reactions as linear pathways is often an oversimplification that does not accurately reflect the complexity of intracellular wiring91.\n\nNetwork analysis indicated NKX3.1-dependent modulation of a series of interconnected functional modules and enabled a tentative framework for the transcriptional program induced by NKX3.1 in human prostate epithelial cells. Broadly speaking, NKX3.1 activation culminates in the downregulation of cellular motility as well as MYC and IFN/STAT activity and in the upregulation of p53 activity, the Notch pathway, and PDGF signaling (Figure 7C). Many of these changes are readily consistent with the tumor suppressor function of NKX3.1 observed in knockout mice3–5.\n\nImportantly, network analysis allowed us to pinpoint several unanticipated pathways on which NKX3.1 appears to impinge. For example, the analysis suggested a major role for TNFα whose mRNA was induced by NKX3.1. TNFα is a well-established inducer of MAP kinase signaling, including JNK and p38 kinases. Significantly, IL1α was also induced by NKX3.1 (Supplementary Table 1) thus further augmenting MAPK activation. JNK activates AP1 transcriptional activity thus readily rationalizing the strong overrepresentation of AP1 binding sites in NKX3.1 responsive genes. Localized NKX3.1-mediated TNFα-JNK signaling in prostate epithelial cells may promote and maintain their differentiation state thus suppressing tumorigenesis. The important role of JNK signaling in cell differentiation is well established92,93. The finding that pro-inflammatory cytokines also destabilize NKX3.1 protein35 indicates a negative feedback loop that may counteract their pro-apoptotic function (Figure 7C).\n\nImportantly, the NKX3.1-induced gene signature is, to a large extent, a mirror image of the gene expression pattern found in early human prostate cancers devoid of NKX3.133. This inverse pattern further suggests that NKX3.1 is a key driver of luminal cell differentiation, whereas loss of NKX3.1 would allow luminal cells to dedifferentiate into a state with higher proliferative capacity thus making them more vulnerable to the acquisition of additional oncogenic events perhaps augmented by concurrent defects in DNA repair. Clearly such additional events are essential for prostate carcinogenesis given that PIN in NKX3.1 knockout mice does not progresses to overt prostate cancer, unless further genetic changes are incurred5–8.\n\n\nData availability\n\nfigshare: NKX3.1 expression and interactions Dataset. Doi: 10.6084/m9.figshare.100206494", "appendix": "Author contributions\n\n\n\nCCY performed the NKX3.1 affinity purifications and the biochemical experiments confirming protein interactions. He also performed validation of microarray data by Q-PCR and immunoblotting. AC prepared NKX3.1 adenoviruses and performed the microarray experiment. CYK assisted with tissue culture and the affinity purifications. LMB performed mass spectrometry of NKX3.1 interacting proteins. RW performed statistical analysis of microarray data and assisted in pathway analysis. DAW conceived the study, performed pathway and network analysis using IPA, and drafted the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by grant W81XWH-04-1-0167 from the Department of Defense Prostate Cancer Research Program to DAW. CCY is the recipient of a Prostate Cancer Training award from the Department of Defense Prostate Cancer Research Program (W81XWH-09-1-0423) and a trainee on the NCI-sponsored T32 Training Grant CA121949. The work was also supported by institutional grants P20 CA132386 and P50 GM085764. The generous support of Jeanne and Gary Herberger during the course of this work is gratefully acknowledged.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe are grateful to Dr. W. Hahn for LH cells and to Dr. C. Kane for continued advice in urologic oncology.\n\n\nSupplementary materials\n\n(A) LNCaP cells were transfected with pFLAG-NKX3.1 plasmid or with the empty pFLAG vector. Total cell lysate (lanes 1 and 2) was absorbed to anti-FLAG resin and eluted with FLAG peptides (lanes 5 and 6). The depleted cell lysate after affinity purification is shown in lanes 3 and 4. Immunoblots were probed with the indicated antibodies. The blot with NKX3.1 shows the overexpressed FLAG-NKX3.1 and the endogenous NKX3.1 protein (middle panel). Actin was used as loading reference. Cropped blot images are shown; see Figure S9 for full images. (B) LNCaP cells were transfected with pFLAG-NKX3.1 plasmid, and FLAG-NKX3.1 was detected by indirect immunofluorescence staining with FLAG or NKX3.1 antibodies.\n\n(A) Differential gene expression 7 and 10 h after NKX3.1 expression in LH cells. Note the overall similarity of gene expression differences between GFP and NKX3.1 expressing LH cells at both time points (7 h and 10 h). (B) \"Volcano Plot\" of differentially expressed genes at the 7 h time point. Features marked in red differed significantly 5-fold between GFP and NKX3.1 expressing samples.\n\nIPA-based rendering of mRNAs contained in the 5× datasets that were previously shown to be regulated by p53.\n\nGreen color indicates upregulation, whereas red color signifies downregulation. The arrows represent the expression edges. Solid arrows indicate agreement between observed expression behavior and the behavior expected in response to activation of PDFGB or TGFβ according to the information contained in the IPA database. The stippled arrows indicate disagreement. Example: PDGF is expected to upregulate HES1. Induction of PDGF by NKX3.1 is therefore consistent with the change in HES1 mRNA (edge is solid red arrow). PDGFB is also expected to upregulate THBS1 (red edge), but NKX3.1 expression leads to suppression of THBS1. Hence the edge is a stippled arrow.\n\n(A) The top panels summarize the datsets: Bioset 1 = 5× dataset of mRNAs affected by NKX3.1 expression; Biogroup 1 = AP1 binding site gene set according to1. The bottom panel illustrates the overlap between Bioset 1 and Biogroup 1 in a Venn diagram (left) and in bar graphs (right). The bar graph shows that most genes containing conserved AP1 binding sites are activated by NKX3.1 expression. The individual genes are indicated in Supplementary Table 1 and Supplementary Table 2. (B) Same as above for serum response factor (SRF).\n\n(A) The top panels summarize the datasets: Bioset 1 = 5× dataset of mRNAs affected by NKX3.1 expression; Bioset 2 = Prostate cancer derived cell lines versus normal prostate epithelial cells2. The bottom panel illustrates the overlap between Bioset 1 and Bioset 2 in a Venn diagram (left) and in bar graphs (right). The bar graph highlights the largely opposite gene expression patterns in the two biosets. The individual genes are indicated in Supplementary Table 1 and Supplementary Table 2.\n\n\nReferences\n\nAbate-Shen C, Shen MM, Gelmann E: Integrating differentiation and cancer: the Nkx3.1 homeobox gene in prostate organogenesis and carcinogenesis. Differentiation. 2008; 76(6): 717–27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShen MM, Abate-Shen C: Roles of the Nkx3.1 homeobox gene in prostate organogenesis and carcinogenesis. Dev Dyn. 2003; 228(4): 767–78. PubMed Abstract \| Publisher Full Text\n\nBhatia-Gaur R, Donjacour AA, Sciavolino PJ, et al.: Roles for Nkx3.1 in prostate development and cancer. Genes Dev. 1999; 13(8): 966–77. PubMed Abstract \| Free Full Text\n\nTanaka M, Komuro I, Inagaki H, et al.: Nkx3.1, a murine homolog of Ddrosophila bagpipe, regulates epithelial ductal branching and proliferation of the prostate and palatine glands. 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Korkmaz", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAuthors have extensively studied the tumor suppressor function of NKX3.1 using multiple gene expression profiling approaches with validations. Eventually, they have demonstrated the NKX3.1 interactome, which revealed a complex pattern of interactions with DNA damage repair proteins including Ku70, PARP1 and XRCC5 in addition to other transcriptional regulators such as ILF2 and BANF1.To perform their research, they have used recognized approaches for the analysis of the gene expression patterns upon ectopic expression of NKX3.1 in immortalized human prostate epithelial cells with a basal phenotype, which revealed a rapid and extensive re-programming with 158 mRNAs changing higher than 5-fold and 331 mRNAs changing higher than 3-fold. Since the data obtained and presented here is consistent with the previous reports, especially Bowen et al. as well as Erbaykent-Tepedelen et al., suggest that the NKX3.1-induced gene signature is similar to the gene expression pattern found in early human prostate cancers. Therefore, the data give insights about the requirement of the NKX3.1 as a key driver of luminal cell differentiation, its loss allows luminal cells to dedifferentiate into a state with higher proliferative capacity leading to the increased genetic heterogenity, perhaps augmented by concurrent defects in DNA damage repair pathways.", "responses": [ { "c_id": "1120", "date": "18 Dec 2014", "name": "Dieter A Wolf", "role": "Author Response F1000Research Advisory Board Member", "response": "Since no specific concerns were raised, we thank the reviewer for his efforts!" } ] }, { "id": "5066", "date": "26 Jun 2014", "name": "Philip D. Anderson", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript presents results from multiple experiments identifying and characterizing the interactome of NKX3.1 in immortalized human prostate epithelium (LH) and human prostate cancer cells (LNCaP). The manuscript is well-researched and well-written. This research helps to fulfil an unmet need by the research community, which is to explain the role of NKX3.1 in the prostate epithelium. In that sense, the results are timely. I have only a few comments, questions or points to the authors that need to be addressed.Explain why there are no error bars in Figure 4A? The authors mentioned that they used the 'affy' package in Bioconductor to preprocess their microarray data. Please indicate the affy package version, Bioconductor version, and R version in the methods. The affy package should be cited in the bibliography with this citation: Gautier, L., Cope, L., Bolstad, B. M., and Irizarry, R. A. 2004. affy---analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 20, 3 (Feb. 2004), 307-315. The authors used a heteroscedastic t-test to infer differences in gene expression in their microarray studies. In the methods, please indicate the multiple testing correction that was applied. In NKX3.1-induced transcriptional program, final paragraph: There are 331 mRNAs changed >= 3-fold. But in Pathway analysis, first paragraph, the 3x dataset is 357 genes. Please explain how these datasets are different? Please indicate the kDa ladder on Figure 3B.", "responses": [ { "c_id": "1121", "date": "18 Dec 2014", "name": "Dieter A Wolf", "role": "Author Response F1000Research Advisory Board Member", "response": "Explain why there are no error bars in Figure 4A?This figure shows p values from the pathway analysis, which do not have variations. The authors mentioned that they used the 'affy' package in Bioconductor to preprocess their microarray data. Please indicate the affy package version, Bioconductor version, and R version in the methods.R-version was 2.10.1 and the bioconductor version 2.5 with the appropriate affy package downloaded by the package manager. The affy package should be cited in the bibliography with this citation: Gautier, L., Cope, L., Bolstad, B. M., and Irizarry, R. A. 2004. affy---analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 20, 3 (Feb. 2004), 307-315.The reference has been included. The authors used a heteroscedastic t-test to infer differences in gene expression in their microarray studies. In the methods, please indicate the multiple testing correction that was applied.Due to the low number of replicates (2 per time point), no correction was applied. Our rationale is that we initially cast a wider net by basing our gene lists on uncorrected p values. The lists are subsequently validated by the orthogonal method of pathway analysis, where we make the assumption that random changes would not manifest as enriched pathways. In NKX3.1-induced transcriptional program, final paragraph: There are 331 mRNAs changed >= 3-fold. But in Pathway analysis, first paragraph, the 3x dataset is 357 genes. Please explain how these datasets are different?We apologize for this error, which was corrected. The 3x dataset of 357 genes was used for the analysis. Please indicate the kDa ladder on Figure 3B.Unfortunately, the original film of this blot which was run in 2007 could not be ascertained. However, Fig 6C shows the expression of GFP and NKX3.1 from the same virus with size markers. The relative sizes of the GFP and NKX3.1 bands are consistent between Fig. 3B and Fig. 6C." } ] } ]	1	https://f1000research.com/articles/3-115
https://f1000research.com/articles/3-156/v1	08 Jul 14	{ "type": "Research Article", "title": "An explanatory evo-devo model for the developmental hourglass", "authors": [ "Saamer Akhshabi", "Shrutii Sarda", "Constantine Dovrolis", "Soojin Yi", "Saamer Akhshabi", "Shrutii Sarda" ], "abstract": "The \"developmental hourglass'' describes a pattern of increasing morphological divergence towards earlier and later embryonic development, separated by a period of significant conservation across distant species (the \"phylotypic stage''). Recent studies have found evidence in support of the hourglass effect at the genomic level. For instance, the phylotypic stage expresses the oldest and most conserved transcriptomes. However, the regulatory mechanism that causes the hourglass pattern remains an open question. Here, we use an evolutionary model of regulatory gene interactions during development to identify the conditions under which the hourglass effect can emerge in a general setting. The model focuses on the hierarchical gene regulatory network that controls the developmental process, and on the evolution of a population under random perturbations in the structure of that network. The model predicts, under fairly general assumptions, the emergence of an hourglass pattern in the structure of a temporal representation of the underlying gene regulatory network. The evolutionary age of the corresponding genes also follows an hourglass pattern, with the oldest genes concentrated at the hourglass waist. The key behind the hourglass effect is that developmental regulators should have an increasingly specific function as development progresses. Analysis of developmental gene expression profiles from Drosophila melanogaster and Arabidopsis thaliana provide consistent results with our theoretical predictions.", "keywords": [ "The evolutionary mechanism of conservation during embryogenesis", "and its connection to the gene regulatory networks that control development", "are fundamental questions in systems biology1–3. Several models have been presented in the context of morphological", "molecular", "and genetic developmental patterns. The most widely discussed model is the “developmental hourglass”", "which places the strongest conservation across species in the “phylotypic stage”. The first observations supporting the hourglass model go back to von Baer when he noticed that there exists a mid-developmental stage in which embryos of different animals look similar4. On the other hand", "the “developmental funnel” model of conservation predicts increasing diversification as development progresses5", "6." ], "content": "Introduction\n\nThe evolutionary mechanism of conservation during embryogenesis, and its connection to the gene regulatory networks that control development, are fundamental questions in systems biology1–3. Several models have been presented in the context of morphological, molecular, and genetic developmental patterns. The most widely discussed model is the “developmental hourglass”, which places the strongest conservation across species in the “phylotypic stage”. The first observations supporting the hourglass model go back to von Baer when he noticed that there exists a mid-developmental stage in which embryos of different animals look similar4. On the other hand, the “developmental funnel” model of conservation predicts increasing diversification as development progresses5,6.\n\nRecently, the hourglass model has come under new light. Multiple studies have observed the hourglass pattern across diverse biological processes, including transcriptome divergence7–11, transcriptome age7,12,13, molecular interaction14, and evolutionary selective constraints10,14,15. Despite these observations the genomic basis and even the existence of the developmental hourglass effect have been the subject of an intense debate1,6,13,16–21. More importantly, the underlying mechanism that can shape the developmental process in the hourglass or funnel forms is still unknown.\n\nWe aim to understand the conditions under which the hourglass effect can emerge in a general setting, based on an abstract model for the evolution of embryonic development. The model focuses on a hierarchical network that represents the temporal “execution” of the underlying Gene Regulatory Network (GRN) during development. Each layer of the network corresponds to a developmental stage. The nodes at each layer represent regulatory genes (i.e., genes encoding transcription factors or signaling molecules) that undergo significant activity change at that corresponding stage. The edges from genes at one layer to genes at the next layer represent regulatory interactions that cause those activity changes. We refer to this hierarchical network as Developmental Gene Execution Network (DGEN) to distinguish it from the corresponding GRN. A DGEN is subject to evolutionary perturbations (e.g., gene deletions, rewiring, duplication) that may be lethal, or that may impede development, for the corresponding organism.\n\nThe model predicts that the evolutionary process shapes the DGENs of a population in the form of an hourglass, under fairly general assumptions. Specifically, the number of genes at each developmental stage follows an hour-glass pattern, with the smallest number at the “waist” of the hourglass. The main condition for the appearance of the hourglass pattern is that the DGEN should gradually get sparser as development progresses, with general-purpose regulatory genes at the earlier developmental stages and highly specialized regulatory genes at the later stages. Another model prediction is that the evolutionary age of DGEN genes also follows an hourglass pattern, with the oldest genes concentrated at the waist.\n\nWe have examined the aforementioned predictions using transcriptome data from the development of Drosophila melanogaster and Arabidopsis thaliana. This data is insufficient to reconstruct the complete DGEN of these species but it allows to estimate the number of genes at each developmental stage, given an activity variation threshold. Under a wide range of this threshold, the inferred DGEN shape follows an hourglass pattern, the waist of that hourglass roughly coincides with the previously reported phylotypic stage for these species, and the age of the corresponding genes follows the predicted hourglass pattern.\n\nAs a first-order approximation, a regulatory gene can be modeled in one of several discrete functional states22. In the simplest case, a regulatory gene can act as a binary switch (“on” and “off”) but in general a gene may have more than two functional states. The transition of a regulatory gene X from one functional state to another is often (but not always) caused by one or more upstream regulators of X that go through a functional state transition before X. We use the term transitioning gene to refer to a regulatory gene that goes through a functional state transition at a given developmental time anywhere at the developing embryo.\n\nA DGEN is a directed and acyclic network; see Figure 1a for an abstract example. The vertical direction refers to developmental time, from the zygote at the top to the developed organism at the bottom. In the horizontal direction we can represent different spatial domains, even though this is not necessary and it is not done in our model. For instance, the zygote at the top of the DGEN would be a single domain, while the organism at the bottom of the DGEN would have the largest number of spatial domains. Development is often approximated (conceptually and experimentally) as a succession of discrete developmental stages. The duration of a developmental stage can be thought of as the typical time that is required for a gene’s functional state transition, and it does not need to be the same for all stages. Each layer of a DGEN refers to a developmental stage, and it includes only the transitioning genes during that stage anywhere in the embryo. The same gene can appear in more than one stage if it goes through several functional state transitions during development. Additionally, a DGEN edge from a gene X at stage l to a gene Y at stage l + 1 implies that the functional transition of X caused the functional transition of Y at the next stage. If gene Y has more than one incoming edge, its functional state transition was caused by the coupled effect of more than one transitioning genes at the earlier stage. Any upstream regulators of Y that remained at the same functional state at stage l are not included in that stage of the DGEN.\n\n(A) The circles denote state-transitioning genes, edges represent directed regulatory interactions, and colored boxes refer to spatial domains that form during development. If regulatory genes become increasingly function-specific as development progresses, the network gradually becomes sparser in that direction. (B) Evolutionary perturbations on a DGEN’s structure: Gene A is deleted (DL), while gene B is rewired (RW) losing an outgoing edge. This RW event causes the regulatory failure (RF) of gene C, which then causes a cascade of five more RF events. This cascade causes developmental failure (DF). Note that the removal of some upstream regulators does not always cause an RF event (e.g., genes regulated by A).\n\nThe sequence of developmental stages is denoted by l=1 … L. The set of transitioning genes at stage l is G(l). A gene g at stage l<L regulates a set of downstream genes at stage l + 1 denoted by D(g) (outgoing edges from g). Similarly, a gene g at stage l>1 is regulated by a set of upstream genes U(g) at stage l – 1 (incoming edges to g). The functional transitions at the first stage are assumed to be caused by regulatory maternal genes that initiate the developmental process.\n\nThe model captures certain aspects of both the developmental process, in the form of a given DGEN for each embryo, and of the evolutionary process, as random perturbations in the structure of individual DGENs in a population. The model does not need to capture the actual functional state transitions or the regulatory input function of each gene. It does capture however the dynamic and stochastic effect of structural network perturbations (gene deletion, rewiring and duplication) on the success of the developmental process, as explained in the following.\n\nSimilar to the Wright-Fisher model, we consider a population of N individuals, each represented by a DGEN. In each generation, individuals reproduce asexually, inheriting the DGEN of their parent. Various evolutionary events can cause structural changes in the DGEN of an individual that may result in “developmental failure”. Such individuals (and their DGENs) are replaced with developmentally successful individuals so that the population size remains constant.\n\nThe model is meant to be as general as possible and so the regulatory interactions between genes of successive stages are determined probabilistically, as follows. Each stage l is assigned a regulatory specificity, or simply specificity s(l) with 0 ≤ s(l) ≤ 1. A gene g at stage l acts as upstream regulator for a gene g′ at stage l + 1 with probability s′(l) = 1 − s(l). So, the specificity of a developmental stage determines how likely it is for regulatory genes of that stage to cause a state transition of the genes at the next stage; a higher specificity decreases that likelihood.\n\nOur major assumption is that the regulatory specificity increases substantially as development progresses. In other words, the DGEN becomes gradually sparser along the developmental time axis, starting with s(1)≈0 and ending with s(L)≈1. This assumption is plausible for the following reasons. First, as development progresses the embryo grows in size forming distinct spatial domains. So, extracellular gene regulation becomes more difficult, especially across different domains. Additionally, as development progresses the transitioning genes become more organ- or tissue-specific, implying that their downstream interactions become sparser. Unfortunately, an empirical investigation of the increasing specificity assumption requires knowledge of the complete DGEN for a given species; this is currently not feasible for even the most well-studied model organisms.\n\nThe DGEN structural changes we consider are gene deletions, gene duplications, and gene rewiring:\n\nDeletions (DL): This event removes a gene from the DGEN, including its incoming and outgoing edges. There are many genetic mechanisms that may cause such events. A DL event deletes each gene of an individual and at each generation with probability PDL.\n\nDuplication (DP): This event creates an identical copy of a gene g with the same downstream and upstream regulators and at the same developmental stage as g. The two genes may have different fates if one of them is subject later to deletion or rewiring. Otherwise, the two genes are considered identical. A DP event duplicates each gene of an individual and at each generation with probability PDP.\n\nRewiring (RW): This event changes the upstream and/or downstream regulators of a gene. Changes in the upstream versus downstream regulators may have different biological basis. The former occur, for instance, as a result of mutations in the transcription factor binding sites in a gene’s promoter or mutations in distal regulatory elements such as enhancers, while the latter may be mostly caused by coding sequence mutations. The details of the rewiring process do not affect the results qualitatively as long as the average density of edges in each stage remains consistent with the specificity of that stage. The specific rewiring mechanisms we use are presented next.\n\nSuppose that a RW event affects gene g at stage l. The upstream regulators of g are recomputed based on the specificity of the previous stage, i.e., by choosing each distinct gene of stage l − 1 with probability s′(l − 1). For the downstream regulators of g, we randomly remove N− existing outgoing edges of g, and then add N+ outgoing edges to randomly chosen genes of stage l + 1 that g is not already connected to. Both N_ and N+ follow a Binomial distribution with \|D(g)\| trials and success probability s′(l). This captures that the downstream regulators of g are derived by incremental changes in D(g), instead of giving g a completely new network configuration (thereby, new regulatory function). The higher the regulatory specificity of a stage, the less likely these incremental changes are. An RW event rewires each gene of an individual and at each generation with probability PRW.\n\nA gene deletion or rewiring event at stage l can remove an upstream regulator from genes at stage l + 1. A loss of incoming edges may trigger the regulatory failure of a gene, as described next.\n\nRegulatory failures (RF): A gene g may not be able to change functional state if some of its upstream regulators U(g) are lost due to DL or RW events (see Figure 1b). Even though regulatory networks are often robust to structural perturbations, even a partial gene loss in U(g) may disable g causing a regulatory failure. It is plausible that the probability of a regulatory failure increases with the fraction of lost upstream regulators. So, if U′(g) is the new set of upstream regulators and \|U(g)\| > \|U′(g)\| > 0, gene g is removed with probability:\n\n\n\nwhile if \|U′(g)\| = 0 we set PRF (1)=1. z is the RF parameter and it depends on the robustness of regulatory interactions to gene loss (see Figure 2).\n\nr is the fraction of upstream regulating edges that are lost due to a DL or RW event.\n\nWhen a DL or RW event causes one or more RF events, the latter can trigger additional RF events in subsequent developmental stages, leading to cascades of regulatory failures. Such RF cascades may cause developmental failure, meaning that the developed embryo is unable to survive or reproduce.\n\nDevelopmental failure (DF): The last stage of a DGEN represents the fully developed embryo. If that stage includes Γ transitioning genes at a successfully developed embryo, the simplest assumption is that an individual with less than Γ genes at stage-L has failed to develop properly. Such DGENs are removed from the population and they are replaced with randomly chosen but successfully developed DGENs. We have also experimented with two variations of the DF event: first, the individual is removed if its last stage has less than Γ − γ genes, where γ is small relative to Γ, and second, the probability of a DF event increases as the number of genes at stage-L decreases below Γ. The qualitative results, as described next, do not change with these two model variations.\n\n\nMethods\n\nHourglass score H. Suppose that w(l) denotes the number of transitioning genes in stage l and let b be the stage with the minimum number of such genes. We construct the sequences X={w(l)}, l = 1, … b} and Y={w(l)}, l = b, … L}. τX and τY denote the normalized univariate Mann-Kendall statistic for monotonic trend in each sequence, respectively (τ is -1 for decreasing, +1 for increasing and almost 0 for random sequences). The H score is defined as H = (τY − τX)/2. See Figure 3 for an illustration, and for the definition of a more robust version of H.\n\nLet w(l) be the width of stage l. Let wb be the minimum width across all stages, and suppose that this minimum occurs at stage l = b; this is the waist of the network (ties are broken so that the waist is closer to ⎣L/2⎦). Consider the sequence X = {w(l)}, l = 1, … b} and the sequence Y = {w(l)}, l = b, … L}. We denote the normalized univariate Mann-Kendall statistic for monotonic trend on the sequences X and Y as τX and τY , respectively. The Mann-Kendall statistic varies between -1 (decreasing) and 1 (increasing), and it is approximately zero for a random sequence. We define H = (τY − τX)/2; H is referred to as the hourglass score. H = 1 if the DGEN is structured as an hourglass, with a decreasing sequence of b stages followed by an increasing sequence of L − b stages. In the computational modeling results, we do not consider the width of the first stage because it can never decrease in Models-1,2,3. We also define a variation of the hourglass score in which we do not take into account adjacent stages in calculating the two Mann-Kendall statistics. That statistic is denoted by H˜ and is referred to as the robust hourglass score.\n\nStatistical analyses. All biological data processing and analyses were performed using custom scripts written in Java (JDK v1.6) [Dataset. 14].\n\nDrosophila data and treatment. Developmental gene expression profiles for D. melanogaster are obtained from two sources. First, we obtained microarray data from Kalinka et al.9 for 3,610 genes. The expression level of each gene is calculated as the median of probes mapping to that gene. Each stage represents a 2-hr interval during the first 20 hours of embryogenesis (10 stages). The second source is RNA-Seq data from Graveley et al.23. Raw data are processed to RPKM values. Each stage represents a 2-hr interval during the first 24 hours (12 stages). Genes with zero RPKM value in all stages are discarded, resulting in 14,110 genes.\n\nArabidopsis data and treatment. Genome-wide expression profiles of a complete developmental series from the zygote to the mature embryo in A. thaliana were obtained from Xiang et al.24. This comprised of microarray expression levels for 25,207 genes across seven developmental stages. Signal background correction and normalization of raw expression values was carried out by Xiang et al.24.\n\nTransitioning gene identification. Suppose that the reported expression value of gene i at stage l is ei,l. We analyze both these “absolute” expression values as well as the normalized expression values, given by e′i,l=ei,l/Σjej,l.\n\nThe identification of transitioning genes follows the same method for both absolute and normalized expression levels. In the case of normalized expressions, we calculate δi,l = e′i,l – e′i,l−1 for each gene and at each stage l=2 … L. Gene i is considered “transitioning” at the stage-pair (l − 1, l) if \|δi,l\| > c, where c is a given transition threshold. This condition is more robust to noise than a ratio-based rule (e′i,l/e′i,l−1) for the identification of transitioning genes. Note that a gene may be transitioning at more than one stage-pair, but it may also not be transitioning at any stage-pair.\n\nTranscriptome age index (TAI). We collected the groups of orthologs for each gene in Drosophila using two databases, OrthoDB25 and OrthoMCL26. The Eumetazoa data were taken from OrthoDB, while Fungi and Plants species were retrieved from OrthoMCL, and the two datasets were merged. Using these orthologs we then assigned an age index to each gene based on its absence and presence in a phylogenetic tree of 24 well-diverged species11,12 [Dataset. 7] (see Figure 4).\n\nEach gene is assigned to one of the following six ages: Level-1: Common ancestor to Fungi, Plants and Eumetazoa. Level-2: Common ancestor to Fungi and Eumetazoa. Level-3: Common ancestor to all Eumetazoa. Level-4: Common ancestor to all Bilateria. Level-5: Common ancestor to all Arthropoda. Level-6: Common ancestor to all Dipteria.\n\nThe transcriptome age index (TAI) values for Arabidopsis genes were obtained from11.\n\nAge index for each stage-pair. Suppose that we identify transitioning genes based on the normalized expression levels, and that n(l) genes are assigned to stage-pair (l − 1, l). Denote by pi the phylogenetic rank (TAI value) of gene i. The age index assigned to that stage-pair is given by TAI(1)=(∑i=1n(l)pie'i,l)/∑i=1n(l)e'i,l. The same method is used when transitioning genes are identified based on absolute expression levels.\n\n\nResults\n\nWe simulate the presented model to examine the properties of the surviving DGENs as evolutionary time progresses. The initial population consists of N identical DGENs with Γ genes at each stage. The edges between genes are constructed probabilistically based on the specificity of each stage, as described previously. Simulating the complete model would not show the significance of individual mechanisms such as the increasing specificity assumption. For this reason we construct a sequence of four models with increasing complexity, presenting results separately for each of them:\n\nModel-1: Constant specificity. Each stage has the same specificity, s(l)=0.5 for l = 1 … L − 1. Further, this model does not include gene deletion and duplication. Gene rewiring can cause RF and DF events even if there are no DL or DP events.\n\nModel-2: Increasing specificity. The difference from Model-1 is that the specificity is gradually increasing across developmental stages. Unless noted otherwise, the specificity is linear, s(l) = l/L for l = 1 … (L − 1); a nonlinear specificity function is also considered, which we describe later.\n\nModel-3: With gene duplications. Model-3 adds DP events in Model-2. The duplication probability PDP is set so that the average size of a DGEN, across the entire population, stays within a given range (70%–80% of L × Γ genes).\n\nModel-4: With gene deletions. Model-4 adds DL events in Model-3 (complete model). The deletion probability PDL is set so that the average size of a DGEN, across the entire population, stays within the same range as in Model-3.\n\nIn Model-1 and Model-2, genes can be only removed (due to RW events, potentially followed by RF cascades) and so the average DGEN size decreases as evolutionary time progresses, which is unrealistic. Model-3 and Model-4 are more realistic because they can maintain a roughly constant DGEN size in the long-term. However, as will be shown next, all aspects of the developmental hourglass effect can already be seen with Model-2 (but not with Model-1). This highlights the increasing specificity assumption as the key property behind the developmental hourglass effect.\n\nHourglass shape. A first observation is that as evolutionary time progresses, DGENs acquire an “hourglass-like” shape in Models-2,3,4. This means that the width of each stage first decreases until a certain stage (referred to as the waist of the hourglass) and then gradually increases. The hourglass may not be symmetric with respect to the waist. To quantify this observation, we define an “hourglass score” H (see Methods and Figure 3) that is equal to 1 if the sequence of L stage widths consists of two segments: a decreasing sub-sequence of k ≥ 2 stages followed by an increasing sub-sequence of L − k ≥ 2 stages. Figure 6a shows the hourglass score for the population of DGENs in Model-2. Similar graphs for the three other models are shown in Figure 5a, Figure 7a, and Figure 8a. The H score quickly increases in the three models that have increasing specificity, and it fluctuates close to 1 afterwards. What is the reason behind the hourglass shape of DGENs? When a gene g is rewired at stage l, it may trigger RF events in stage l + 1 depending on the number of its lost outgoing edges. In the first few stages, where specificity is low, it is unlikely that a gene would lose a large fraction of its (typically many) incoming edges. In the last few stages, where specificity is high, edges are unlikely to get rewired in the first place. In the mid-stages however, where the specificity is close to 50%, there is higher variability in the number of outgoing edges lost or gained due to RW events. The loss of several outgoing edges due to an RW event at stage l can trigger several RF events and gene removals in the subsequent stage. Thus, the probability of RF events in mid-stages is higher than in early/late stages, making the removal of genes more likely in the former.\n\nParameters: 10 runs with different initial populations, N=10 individuals, L=10 stages, specificity function s(l)=0.5 for all stages, Γ=100 genes at each stage initially, RF parameter z=4, 1,000,000 generations, probability of RW event PRW =10−4. The red line is the median and the green boxes are the 10th, 25th, 75th, and 90th percentiles, across all individuals and all runs. (a) The hourglass score H across evolutionary time. (b) Lethality probability at each stage. (c) Age of existing genes at the last generation.\n\nParameters: 10 runs with different initial populations, N=1000 individuals, L=10 stages, specificity function s(l)=l/L (l=1 … L − 1), Γ=100 genes at each stage initially, RF parameter z=4, 500,000 generations, probability of RW event PRW=10−4. The red line is the median and the green boxes are the 10th, 25th, 75th, and 90th percentiles, across all individuals and all runs. (A) The hourglass score H across evolutionary time. (B) Lethality probability at each stage. (C) Age of existing genes at the last generation.\n\nParameters: 10 runs with different initial populations, N=10 individuals, L=10 stages, specificity function s(l)=l/L (l=1 … L − 1), Γ=100 genes at each stage initially, RF parameter z=4, 1,000,000 generations, probability of RW event PRW = 10−4. The probability of gene duplication PDP is adjusted dynamically so that the average DGEN size stays between 700 and 800 genes. The red line is the median and the green boxes are the 10th, 25th, 75th, and 90th percentiles, across all individuals and all runs. (a)The hourglass score H across evolutionary time. (b) Lethality probability at each stage. (c) Age of existing genes at the last generation.\n\nParameters: 10 runs with different initial populations, N=10 individuals, L=10 stages, specificity function s(l)=l/L (l=1 … L − 1), Γ=100 genes at each stage initially, RF parameter z=4, 1,000,000 generations, probability of RW event PRW = 10−4. The probability of gene duplication PDP is adjusted dynamically so that the average DGEN size stays between 700 and 800 genes. The probability of gene deletion (DL) is PDL = 10−6. The red line is the median and the green boxes are the 10th, 25th, 75th, and 90th percentiles, across all individuals and all runs. (a) The hourglass score H across evolutionary time. (b) Lethality probability at each stage. (c) Age of existing genes at the last generation.\n\nThe constant specificity of Model-1 does not result in an hourglass pattern [Dataset. 1] (see Figure 5a) for the following reason. RW events at stage l can cause RF events at the next stage with the same probability, independent of l. However, after the occurrence of an RF event, the size of the potential cascade increases as l decreases simply because there are more subsequent stages to affect. This gives DGENs a “funnel-like” shape with a gradually increasing number of genes after stage-1; H fluctuates around 0.5, as expected for an increasing sequence.\n\nStage lethality. Another aspect of the developmental hourglass is in terms of the significance of each stage for the survival of the embryo. We define lethality of stage l as the probability that a RW or DL event at stage l starts a RF cascade that eventually leads to a DF event. We estimate this probability at generation i as the fraction of RW and DL events, during the past i generations, that occurred at stage l and led to a DF.\n\nIn Model-1, there is no clear trend for the stage lethality probability (see Figure 5b); with the exception of the last stage (in which RW events cannot result in gene loss), the lethality probability is roughly the same at all stages. In the three models with increasing specificity, however, we observe a clear pattern: the lethality gradually increases until the waist of the hourglass, and then it decreases [Dataset. 2, Dataset. 3 and Dataset. 4] (see Figure 6b, Figure 7b, and Figure 8b). The reason, as explained earlier, is that the probability of RF events in mid-stages is higher that in early/late stages. Additionally, after the formation of the hourglass shape the mid-stages have relatively few genes and so an RF event in those genes is more likely to initiate a lethal RF cascade.\n\nAge of genes. A third aspect of the developmental hourglass effect is related to the evolutionary age of genes. The age of a gene g at generation i is defined as A(g) = i − t0(g), where t0(g) is the generation at which g was most recently rewired (and 0, if it was not rewired earlier). The rationale behind this definition is that a rewiring event may give that gene a new function, at least in terms of its upstream and downstream regulators.\n\nIn the case of Model-2, Figure 6c shows the median age of the genes at each stage, considering the population of all genes across all individuals at a given generation. See Figure 5c, Figure 7c, and Figure 8c for the same results with the three other models. The evolutionary age at stage l follows the same pattern as the lethality probability: it gradually increases until we reach the waist of the hourglass, and then it gradually decreases. Genes at intermediate stages tend to be older because, as explained earlier, they are fewer and their rewiring is more likely to be lethal. When one of those genes g is rewired or deleted from a DGEN, the corresponding individual is often replaced (DF event) by another individual that has the same gene g. So, the genes at the waist of a DGEN tend to be more conserved than genes at earlier or later stages.\n\nLocation of waist. What controls the location of the hourglass waist in the developmental process?\n\nThe location of the waist is mostly affected by two parameters of the model: the shape of the specificity function and the RF parameter z. To examine the effect of the former we use the nonlinear function shown in Figure 9. γ is the stage at which the specificity is 50%, and so that stage has the maximum variance in the number of outgoing regulatory edges. RW events at this stage can cause the largest extent of rewiring and so, the highest likelihood of RFs in genes of the next stage. Figure 10a shows that the location of the hourglass waist is “pushed” towards stage γ, even though it does not coincide always with that stage. Parameter z controls the shape of the RF probability: increasing z makes RF events more likely, also increasing the likelihood of lethal RF cascades. Figure 10b shows that as we increase z the hourglass waist moves towards later developmental stages [Dataset. 5].\n\nThis function allows us to control the stage γ at which the specificity is 50%.\n\nThe first is the specificity function. To examine its effect, we use a sigmoid-like mathematical function that controls the stage γ at which the specificity is 50% (see Figure 9). This is the stage with the maximum variance in the number of outgoing regulatory edges. RW events at this stage can cause the largest extent of rewiring and so, the highest likelihood of RFs in genes of the next stage. Graph (a) shows that the location of the hourglass waist is “pushed” towards stage γ, even though it is not always exactly at that stage. The second way to affect the location of the hourglass waist is the parameter z that controls the shape of the RF probability. Increasing z makes RF events more likely, also increasing the likelihood of lethal RF cascades. Graph (b) shows that as we increase z the hourglass waist moves towards later developmental stages. These results are obtained using Model-2. Parameters: 10 runs with different initial populations, N=1000 individuals, L=10 stages, specificity function s(l)=l/L (l=1 … L − 1), Γ=100 genes at each stage initially, RF parameter z=4, 500,000 generations, probability of RW event PRW = 10−4. The graphs show the median (red lines) and the 10th, 25th, 75th, and 90th percentiles (green boxes) of the location of the waist.\n\nGene prevalence. We introduce a “gene prevalence” metric for gene g at time t as the fraction of individuals that include g at evolutionary time t [Dataset. 6]. Figure 11a shows the gene prevalence metric across developmental stages after 500,000 generations, while Figure 11b shows the relation between gene prevalence and gene age. The genes at the waist of the developmental hourglass are not only the oldest but also the most prevalent across the population. The implication of this simulation result is that we can expect that those genes that are transitioning near the waist of the developmental hourglass will be the most conserved genes in a population.\n\nThese results are obtained using Model-2. Parameters: 10 runs with different initial populations, N=1000 individuals, L=10 stages, specificity function s(l)=l/L (l=1 … L−1), Γ=100 genes at each stage initially, RF parameter z=4, 500,000 generations, probability of RW event PRW = 10−4. The graphs show the median (red lines) and the 10th, 25th, 75th, and 90th percentiles (green boxes) for: (a) prevalence of genes in each stage after 500,000 generations, and (b) gene age as a function of gene prevalence. As expected, older genes tend to be more prevalent in the population.\n\nWe have examined the predictions of the previous model using transcriptome data for Drosophila melanogaster and Arabidopsis thaliana [Dataset. 14]. We summarize results from both species here; the corresponding figures for which are Figure 12 and Figure 13 [Dataset. 8 and Dataset. 9]. For Drosophila, we analyze Microarray9 and RNA-Seq23 temporal expression profiles during the first 20 hours of development, taken at 10 stages of 2-hr intervals. We examine whether a) the number of transitioning genes follows an hourglass pattern, b) the waist of that hourglass coincides with the Drosophila phylotypic stage, and c) the evolutionary age of the transitioning genes follows a similar hourglass pattern. The two datasets are described in more detail in the Methods section. With such limited data, we cannot infer the regulatory edges between transitioning genes and we cannot reconstruct the underlying DGEN. However, we can identify the transitioning genes at each developmental stage given a “transition threshold” c (see Methods). Even though the correct value of this threshold is not known, the following results are robust in a wide interval of c, which includes most of the expression variation range across successive developmental stages (see Figure S3 for the CDFs of expression level variations across successive stages [Dataset. 12]).\n\nGraphs (A) and (B) show the hourglass score (normal and robust) as a function of the transition threshold c for the two datasets. Graphs (C) and (D) show the location of the hourglass waist (stage-pair) as a function of the transition threshold c for the two datasets. Graphs (E) and (F) show the Transcriptome Age Index of transitioning genes for three different values of c (chosen so that the number of genes with known age index assigned to each stage is at least 25) for the two datasets. Graph (G) shows the transitioning genes for the Microarray dataset with c=0.0005. The transitioning genes constitute 11% of all genes in that dataset. 53% of those genes transition in a single stage-pair. Of the remaining, 64% transition only in consecutive stage-pairs. Note that if a gene transitions n times, it is counted in n stage-pairs. Similarly, graph (H) shows the transitioning genes for the RNA-Seq dataset with c=0.00025. The transitioning genes constitute 5% of all genes in that dataset. 45% of those genes transition in a single stage-pair. Of the remaining, 52% transition only in consecutive stage-pairs.\n\nGraph (a) shows the hourglass score (normal and robust) as a function of the transition threshold c. Graph (b) shows the location of the hourglass waist (stage-pair) as a function of the transition threshold c. Graph (c) shows the Transcriptome Age Index of transitioning genes for five different values of c (chosen so that the number of genes with known age index assigned to each stage-pair is at least 290). Graph (d) shows the CDFs of the expression level absolute variations \|δ\| across successive stage-pairs. Note that in this case the hourglass waist (in terms of number of transitioning genes) appears in stage-pair (3,4), while the oldest genes appear in the next stage-pair. Graph (e) shows the transitioning genes with c=0.0001. The transitioning genes constitute 7% of all genes in that dataset. 49% of those genes transition in a single stage-pair. Of the remaining, 36% transition only in consecutive stage-pairs.\n\nFigure 12a and Figure 12b show the hourglass resemblance score H (and its more robust variant) as function of c. Note that the H score is close to 1 for a wide range of c, confirming the presence of an hourglass-like structure in terms of the number of transitioning genes. Figure 12g and Figure 12h exhibit this pattern more clearly in the number of transitioning genes for a specific value of c. The two datasets also show reasonable agreement in terms of the assignment of transitioning genes to stage-pairs [Dataset. 10] (see Figure S1).\n\nSecond, the location of the waist in this hourglass pattern, shown in Figure 12c and Figure 12d, occurs at the stage-pair (3,4) or (4,5), depending on c. This is roughly 8 hours after the formation of the zygote, and it includes the phylotypic stage for Drosophila melanogaster9.\n\nWe have also estimated the evolutionary age of most of the transitioning genes at each developmental stage-pair using the Transcriptome Age Index (TAI) metric12 (see Methods). TAI is lower for older genes. Figure 12e and Figure 12f show the average TAI for transitioning genes, weighted by the expression level of each gene, at each stage-pair and for each dataset using three values of c. The TAI index follows the pattern that the model predicts, with older genes (lower TAI values) close to the waist of the hourglass. This result appears consistent with the main observation of Domazet-Loso and Tautz12, even though that study did not analyze transitioning genes.\n\nThe same approach was extended using transcriptome data24 and TAI profiles12 from Arabidopsis. The results obtained from the analysis (Figure 13) were consistent with the predictions of the model, as well as the results obtained from Drosophila data.\n\n\nDiscussion\n\nEarly studies of the developmental hourglass effect mostly analyzed morphological and phenotypic similarities across species2,27. Recently, the focus has shifted towards genomic and molecular comparative studies8,9,11,12,19 that investigate conservation of gene expression variation, sequence conservation, selective constraint on coding sequences, and evolutionary gene “age”. These studies often report contradicting observations: some support strong conservation in earlier developmental stages5,6,19, while others support that strongest conservation occurs at a mid-developmental stage7–15. Nevertheless, the fact that the hourglass effect is observed in highly divergent species across deep phylogenetic scales (including fish, flies and plants), suggests that this observed pattern of conservation may stem from fundamental organization principles.\n\nWhat these principles are has remained elusive. Earlier stages may be conserved because any changes therein could have large cascading effects in later stages5,28,29. Later stages may experience less constraint because as development progresses gene interactions become more modular, and so it is plausible that perturbations there have only local effects1. We refer to them as the “temporal” constraint model and the “spatial” constraint model, respectively, following Tian et al.30.\n\nIn this paper, we developed an evolutionary model of development that combines some aspects of the previous two models. Regulatory perturbations at a certain stage can cause cascades of regulatory failures at subsequent stages (temporal model), while the likelihood that a gene regulates genes at a subsequent stage decreases as development progresses (spatial model). Our computational results lead to the following testable predictions: a) the number of transitioning genes during development follows an hourglass pattern, b) the evolutionary age of the transitioning genes also follows an hourglass pattern, with the oldest genes being at the waist of the hourglass, and c) the genes at the waist of that hourglass are the most essential, in the sense that their deletion maximizes the probability of developmental failure. We have relied on developmental gene expression profiles of Drosophila melanogaster and Arabidopsis thaliana to examine the predictions of the model. The analysis of that data agrees with the first two theoretical model predictions. The increased conservation of genes at the waist provides an indirect confirmation of the model’s third prediction, regarding the essentiality of different genes. Further, our simulations confirm that the details of these regulatory perturbations, such as the probabilities of gene duplication and deletion or the parameter z in the regulatory failure probability, do not affect the results of the model, at least at the qualitative level.\n\nThe use of DGENs in this work was only as an abstract tool to study the effect of gene regulatory perturbations in the developmental process. In future work, it is important to infer the actual DGEN of model organisms. This will require information about gene regulatory interactions across time and space, but it should be possible for at least some developmentally well studied species22. Such DGENs would help to identify the specific genes that form the hourglass waist and their function. Additionally, an inferred DGEN would allow to directly test the increasing specificity assumption.\n\nFinally, we note that the hourglass effect (sometimes referred to as the ”bow-tie” effect) has also been observed in other complex biological and technological systems that exhibit hierarchical modularity and that are subject to evolutionary pressure or optimization tradeoffs31–34. For instance, the Internet “protocol stack” is organized in an hourglass structure35; this pattern was not designed but it emerged through the competition between protocols that serve roughly the same function at each communication layer, during the last 30–40 years. In earlier work, we proposed an abstract model (EvoArch) that captures the evolution of protocol architectures and that predicts the emergence of an hourglass structure. Interestingly, both EvoArch and the model of this paper share the same principle: the underlying hierarchical networks that control both systems should be increasingly sparser as complexity increases, i.e., the specificity of each complexity stage (or layer) should be increasing. In the future, we will further investigate this common organization principle between biological and technological systems.\n\n\nData and software availability\n\nZENODO: An evo-devo model for the developmental hourglass: code and data, doi: 10.5281/zenodo.1057936\n\nLicense: GNU GPLv3", "appendix": "Author contributions\n\n\n\nS.A., S.S., C.D., and S.Y designed research; S.A. and C.D. developed the computational model; S.A. and S.S. analyzed data; and S.A., S.S., C.D., and S.Y wrote the paper.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the National Science Foundation (Grant No. 0831848 to C.D.). Any opinions, findings and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation (NSF).\n\n\nAcknowledgements\n\nWe thank Alex Kalinka for providing time-series gene expression data for Drosophila melanogaster. We also thank Joshua Weitz, Sridhar Hannenhalli and Todd Streelman for helpful comments on the manuscript.\n\n\nNotes\n\nDedicated to the memory of the first author, Saamer Akhshabi, who passed away on March 6, 2014.\n\n\nSupporting information\n\nBecause the appropriate transition threshold may be different at each dataset, we use a different threshold for each dataset, say c1 and c2. For each pair (c1, c2), we determine the transitioning genes at each stage-pair with the corresponding dataset (i.e., L − 1 pairs of gene sets), and then calculate the average Jaccard similarity across these L − 1 pairs. The Jaccard similarity maps show this average across all stage-pairs for various threshold pairs (c1, c2). In graph (a) with normalized expression levels, when the two thresholds are roughly equal, the average Jaccard similarity is as high as 50%; this means that about 2/3 of the genes assigned to a certain stage-pair using one dataset are also assigned to the same stage-pair using the other dataset. Graph (b) shows a similar Jaccard similarity map for the case of absolute expression levels. [Dataset. 10].\n\nGraphs (a) and (b) show the hourglass score (normal and robust) as a function of the transition threshold c for the two datasets. Graphs (c) and (d) show the location of the hourglass waist (stage-pair) as a function of the transition threshold c for the two datasets. Graphs (e) and (f) show the Transcriptome Age Index of transitioning genes for three different values of c (chosen so that the number of genes with known age index assigned to each stage is at least 10) for the two datasets. Graph (g) shows the transitioning genes for the Microarray dataset with c=5000. The transitioning genes constitute 21% of all genes in that dataset. 62% of those genes transition in a single stage-pair. Of the remaining, 58% transition only in consecutive stage-pairs. Similarly, graph (h) shows the transitioning genes for the RNA-Seq dataset with c=10000. The transitioning genes constitute 5% of all genes in that dataset. 45% of those genes transition in a single stage-pair. Of the remaining, 53% transition only in consecutive stage-pairs. [Dataset. 11].\n\n(a) normalized expressions, Microarray, (b) normalized expressions, RNA-Seq, (c) absolute expressions, Microarray, (d) absolute expressions, RNA-Seq. [Dataset. 12].\n\nGraph (a) shows the hourglass score (normal and robust) as a function of the transition threshold c. Graph (b) shows the location of the hourglass waist (stage-pair) as a function of the transition threshold c. Graph (c) shows the Transcriptome Age Index of transitioning genes for five different values of c (chosen so that the number of genes with known age index assigned to each stage-pair is at least 170). Graph (d) shows the CDFs of the expression level absolute variations \|δ\| across successive stage-pairs. Graph (e) shows the transitioning genes with c=5000. The transitioning genes constitute 8% of all genes in that dataset. 48% of those genes transition in a single stage-pair. Of the remaining, 38% transition only in consecutive stage-pairs. [Dataset. 13].\n\n\nReferences\n\nRaff RA: The shape of life: genes, development, and the evolution of animal form. University of Chicago Press. Trends Ecol Evol. 1996; 11(10): 441–442. 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Data Source" }	[ { "id": "5563", "date": "24 Jul 2014", "name": "Marcel Quint", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study by Akhshabi et al. aims to provide the community with an explanatory theoretical model for the developmental hourglass phenomenon. Motivated by the question concerning the regulatory mechanism causing the developmental hourglass pattern during embryogenesis, the authors developed an evolutionary model of regulatory gene interactions during development to identify the conditions under which the molecular hourglass effect might emerge in general. For this purpose their evolutionary model focuses on hierarchical gene regulatory networks that control the corresponding developmental processes. Based on the output of the model the authors predict the emergence of an hourglass pattern in the structure of a temporal representation of the underlying gene regulatory network and correlate this effect with the evolution of protocol architecture found for the Internet’s “protocol stack”. Based on this universal finding the authors speculate that the developmental hourglass pattern might be a causal effect of a common organization principle during the establishment of complexity and is based on the underlying hierarchical network structure. The paper is very interesting, well written, and will potentially help the community to better understand the developmental hourglass phenomenon. However, we see some issues with the model assumptions and strongly suggest including the following aspects in a revised version. Summary of the points that have to be addressed: Include a clear reference and paragraph to Raff's hypothesis and how it influenced the modeling of DGEN.Correlate and reference major assumptions of the DGEN model that seem plausible with findings from experimental studies that validate this plausibility.Correlate the output of the DGEN simulation with existing findings of studies addressing similar questions of the causality of the developmental hourglass phenomenon and discuss potential simplifications of the model assumptions that do not fit experimental results or that are not derivable via experiments yet.Include a clear statement how the exact values of the stage specificity (regulatory specificity) s(l) have been derived.State more clearly that s(l) is the crucial parameter determining the observed phenomena.Correct the TAI formula.Explain why merging OrthoDB results with OrthoMCL results might be plausible and why comparing it with results obtained from phylostratigraphy is reliable.Discuss more clearly how the DGEN model can help to find causal processes of the developmental hourglass phenomenon based on recent knowledge: the three predictions of the DGEN model a) – c) mentioned in the Discussion section seem not integrative enough to allow future studies to correlate existing results provided by the community with the predictions obtained by the DGEN model.Specific comments to the separate sections:In general, most of our criticisms concern the model description and the methods section. Both are integral to the validity of the obtained results, which are nicely reported and discussed. Introduction Modeling the evolution of embryonic development as directed acyclic graph (DAG) in which the nodes correspond to state-transitioning genes and the edges model directed regulatory interactions causing significant activity change at the corresponding stage seems plausible. The authors refer to this model as DGEN. A testable hypothesis proposed by the authors is that if regulatory genes become increasingly function-specific as development progresses, the network gradually should become sparser in that direction and evolutionary older genes should be concentrated at the waist of the hourglass (Figure 1). This hypothesis is essential to detect possible regulatory mechanisms that cause the developmental hourglass pattern. We therefore suggest including references to existing biological studies investigating the phenomenon of morphological complexity being correlated with regulatory specialization to support the validity and plausibility of their DGEN model assumptions. A biological motivation of this hypothesis comes from Rudolf A. Raff who proposed that early embryogenesis is governed by global pattern formation processes and that during later development, the embryo is being organized into developmental modules, allowing each module to differentiate autonomously. Raff furthermore proposed that the phylotypic stage marks the transition from early global development to the modular mode later in development1. The authors do cite Raff in the first sentence of their introduction, but should state more clearly in which manner their model was inspired by Raff's hypothesis. Numerous studies investigated the phenomenon that increasing morphological complexity is correlated with increasing modularity and specialization of the underlying regulatory apparatus 2. Here the authors predict that the main condition leading to the appearance of the hourglass pattern is that the DGEN should gradually get sparser as development progresses, with general-purpose regulatory genes at the earlier developmental stages and highly specialized regulatory genes at the later stages, expressing the oldest genes during mid development. We suggest to also include findings investigating the functionality and age of the genes that have been shown to be active during mid embryogenesis to correlate potential outcomes of the DGEN prediction that evolutionary older genes should be concentrated at the waist of the hourglass with results obtained by recent studies 3 4 5 6 7 8 9 10 11 12. These aforementioned studies were able to show that during mid embryogenesis evolutionary old genes or evolutionary old processes that are shared within and between phyla are most active. In our opinion it would be very interesting to test the degree of intersection between genes predicted to be causal for the waist of the DGEN hourglass and evolutionary conserved genes that have been found to be expressed during mid embryogenesis. We are aware that the model uses the number of genes at each developmental stage combined with the age of the corresponding genes to observe constraints during development. Hence, only the authors can estimate how much effort would be needed to identify such intersecting genes. If the search for intersecting genes returned by the DGEN model during mid development and genes found by previous studies should be possible, the authors could integrate a motivation section to find these intersecting genes within the Introduction section. Model description The model description is quite intuitive. However, as the validity of the whole study depends on the model parameters chosen, several points need clarification including the rationale and motivation to choose exactly these parameters. One of the most crucial parameters of the model is the regulatory specificity s(l), with 0 <= s(l) <= 1. We think, that adding a more detailed paragraph discussing how s(l) is being estimated, would be highly beneficial due to the importance for the parameter s(l) for the entire model and predictions. In Model-1-4 the authors choose three different values for s(l): In Model-1: s(l) = 0.5 and in Model-2-4: s(l) = l/L, and in Figure 9 a non-linear function s(l) = 0.9 – (0.8 / 1 + exp(\\gamma - l) ). A gene g at stage l is modeled to act as upstream regulator for a gene g’ at stage l + 1 with probability s’(l) = 1 – s(l). From this follows that each potential upstream regulator in stage l has the same probability s’(l) to regulate genes g’ in stage l + 1. In other words, Model-1 assigns each potential upstream regulator with the same probability s’(l) = 1 – 0.5 = 0.5 to regulate genes g’ in stage l + 1, in Model-2: s’(l) = 1 – l/L = const., and for the non-linear function s(l): s’(l) = 1 – (0.9 – (0.8 / 1 + exp(\\gamma - l) )) = const. It would be advantageous, if the authors could include motivations for the three cut-offs, especially how they derived the sigmoid-like mathematical function (Figure 10, legend caption) they later denote as non-linear function s(l). We do see that modeling the stage specificity with a constant value, an increasing value, and a non-linear function is plausible, but a clear motivation or derivation of the exact values would be beneficial for a better understanding of the modeling process. For the probabilities PDL and PDP the explicit formula to compute the corresponding probabilities could also be included within the paragraph analogous to PRF. This would enable a clearer reproducibility. The authors discuss that their major assumption is that the regulatory specificity increases substantially as development progresses, hence the DGEN becomes gradually sparser along the developmental time axis starting with s(1) ~ 0, … , s(L) ~ 1. Here the authors should include references to experimental observations that support their assumption. Since Models 2-4 rely on this assumption, published experimental studies in line with this assumption need to be referenced. Otherwise, the models applied would seem largely academic. Methods Hourglass score HHere w(l) is defined as the number of transitioning genes in stage l. In Figure 3 and in section 'Hourglass shape' w(l) is referred to as stage width. In case the definition of w(l) is equivalent to the stage width, it would be beneficial for the reader to see the term stage width in the same sentence as the initial definition of w(l). A correct understanding of the stage width is important to understand the measurement of H.Furthermore, the univariate Mann-Kendall statistic should be referenced to allow a non-statistical community to better understand this way of statistical modeling. Transcriptome age index (TAI)In this section the authors describe and perform two different methods to assign each gene a corresponding evolutionary age. For D. melanogaster they collected groups of orthologs from OrthoDB and OrthoMCL, whereas for A. thaliana they obtained phylostratum assignments from Quint et al.10 that was derived by phylostratigraphy[ref-13.]We do not understand the motivation of merging orthologous gene groups obtained from OrthoDB and OrthoMCL, as both approaches apply different parameters and heuristics to determine orthologous genes. Was this done to maximize the number of potential orthologs to be included in the study? In addition, why did the authors use a different approach for the plant gene set? This seems somewhat inconsistent and statement concerning the plausibility to compare results returned by the DGEN model based on age assignments obtained from phylostratigraphy (A. thaliana) with OrthoDB and OrthoMCL merged orthologs (D. melanogaster) would be appreciated. Furthermore, the database specific parameters they used in OrthoDB and OrthoMCL should be mentioned as well as the database version they used, e.g. OrthoDB2, or OrthoDB3, … , or OrthoDB6. Age index for each stage-pairThere seems to be a typo within the TAI formula. As defined by Domazet-Loso and Tautz6 and adapted to the author’s notation the formula of the TAI should begin TAI(l) not TAI(1). Results The authors show based on their findings returned by Model-2-4 that the increasing specificity assumption is the key property behind the developmental hourglass effect. We think that this statement is not clear enough. The authors could phrase more clearly what they mean by “key property behind the hourglass effect” inferred from Model-2-4. Age of genes Here the authors motivate that defining the age of a gene as A(g) = i – t0(g) is plausible, because a rewiring event may give a gene a new function, at least in terms of its upstream and downstream regulators. This statement needs validation from the literature and from experimental studies that already tested this hypothesis for specific rewiring events. Furthermore, the authors show (Figure 8c) that the evolutionary age at stage l follows the same pattern as the lethality probability: gradually increasing until the waist of the hourglass, and then gradually decreasing. This finding should be correlated with the findings by Galis and Metz3 and should be discussed more clearly in the Discussion. In the first sentence of the last paragraph before the Discussion, \"… and TAI profiles from Arabidopsis\" should be referenced to reference 11 and not 12. Discussion The authors discuss that the observed pattern of conservation (developmental hourglass) may stem from fundamental organization principles and that the exact origin of these principles remains elusive. Here the authors should include existing studies that also aim at predicting a universal organization principle during development (modularization, etc.) and what exact problems are preventing studies to elucidate this universal phenomenon. We do note, that the authors cite studies that aim to explain the effects causing the early phase of the developmental hourglass 141516 and studies aiming to explain effects causing the late phase of the developmental hourglass1, but we think that it should be explicitly stated that the model is designed to test aspects of Raff’s initial hypothesis and that also more recent studies have been investigating the underlying reasons for the pattern in the early phase of the hourglass17.", "responses": [ { "c_id": "1122", "date": "18 Dec 2014", "name": "Constantine Dovrolis", "role": "Author Response", "response": "First, we would like to thank the two authors of this review for their very thorough reading of our paper. Their comments and suggestions have been extremely helpful in revising the paper (please look at Version-2). We would also like to apologize for not revising the paper earlier.Review: “Modeling the evolution of embryonic development as directed acyclic graph (DAG) in which the nodes correspond to state-transitioning genes and the edges model directed regulatory interactions causing significant activity change at the corresponding stage seems plausible. The authors refer to this model as DGEN. A testable hypothesis proposed by the authors is that if regulatory genes become increasingly function-specific as development progresses, the network gradually should become sparser in that direction and evolutionary older genes should be concentrated at the waist of the hourglass (Figure 1). This hypothesis is essential to detect possible regulatory mechanisms that cause the developmental hourglass pattern. We therefore suggest including references to existing biological studies investigating the phenomenon of morphological complexity being correlated with regulatory specialization to support the validity and plausibility of their DGEN model assumptions. A biological motivation of this hypothesis comes from Rudolf A. Raff who proposed that early embryogenesis is governed by global pattern formation processes and that during later development, the embryo is being organized into developmental modules, allowing each module to differentiate autonomously. Raff furthermore proposed that the phylotypic stage marks the transition from early global development to the modular mode later in development.”Summary - Point#1: “The authors do cite Raff in the first sentence of their introduction, but should state more clearly in which manner their model was inspired by Raff's hypothesis.”Summary - Point#2: “Correlate and reference major assumptions of the DGEN model that seem plausible with findings from experimental studies that validate this plausibility.”Response to Point #1: The reviewers are right to highlight the connection between our “increasing regulatory specificity” assumption and Raff’s hypothesis. We have included a paragraph in the Discussion section to put Raff’s hypothesis in the context of this work.A first remark is that our assumption is consistent with Raff’s basic premise that modularity increases as the embryo develops. Second, Raff’s hypothesis (see chapter-6 of [1], and in particular Figures 6.6 and 6.7) also states that the “level of interaction” or the “interconnectivity between (body) elements” is maximized at the phylotypic stage. This may be viewed initially as a contradiction between Raff’s hypothesis and our increasing specificity assumption, which states that the density of regulatory interactions is maximized at the earliest stages of development. Note however that Raff’s hypothesis was not stated in terms of gene regulatory interactions -- he was referring more generally to “developmental flexibility”, arguing that early development is flexible because it governs robust and general global patterning processes, late development is also flexible because “signaling events within the primordia are little influenced by events in other primordia”, while mid-development (phylotypic stage) is least flexible because of the “high interconnectivity between elements that will later come to represent separate modules.” If we think of “developmental flexibility” as the ability of an embryo to survive gene mutations and rewiring at different stages of the developmental process, Raff’s hypothesis is actually consistent with our results regarding the “lethality probability” at each developmental stage (see Figs 6-B, 7-B amd 8-B). The lethality probability is maximized at the phylotypic stage, and it is significantly lower at early and late developmental stages, following the same pattern with Raff’s “developmental flexibility”.Response to Point #2: First, to the extent of our knowledge there is no prior work that directly validates the assumption of increasing regulatory specificity as formulated in our paper (i.e., with the specificity of a developmental stage defined as the topological density of regulatory edges in that stage). We are planning to examine the validity of this assumption in the future using various approaches:First we plan to analyze the aforementioned developmental GRNs for the sea-urchin that have been directly examined by Dr. Davidson’s lab at Caltech.Second, we are in the process of examining other genomic metrics, which can be viewed as “proxies” to regulatory specificity. For example, we have defined new measures to quantify how specific a gene’s expression profile in a developmental stage is compared to other developmental stages, which we have tentatively named as “developmental stage specificity index (DSI)”. Similarly, we defined “tissue specificity index (TSI)”, measuring the bias of a gene’s expression in a specific tissue compared to many other tissues. Analyses of these measures using genomic data from Drosophila so far have revealed generally increasing trends, which are consistent with the idea of increasing specificity. However we are still working on generalizing these analytical tools to data sets generated by different methods and from different species. Importantly, our model focuses DGENs underlying development, and genomic data can obscure the true signals from genes constituting DGENs. Thus we are also working on examining these specificity measures using inferred DGENs. Additionally, we are developing other methods to approximate functional specificity. We hope to complete these analyses as a follow-up paper. The current paper provides theoretical motivation.Third, as the reviewers also point out, there is a large body of prior work in developmental biology that confirms the increasing modularity in the developing embryo (for instance, see Wagner et al., 2007) as well as the increasing specificity of the developmental process at the signaling or genomic level. The connections between this pattern of increasing modularity and the structure of the underlying gene regulatory networks are still not well understood however. We think that it would be misleading if we had argued that these modularity patterns provide a direct validation for our “increasing regulatory specificity” assumption. Please note that we have cited the paper by Wagner et al. at the Introduction and Model Description sections, providing some connections between this work and earlier work on the role of modularity in developmental biology. Review: “Numerous studies investigated the phenomenon that increasing morphological complexity is correlated with increasing modularity and specialization of the underlying regulatory apparatus 2. Here the authors predict that the main condition leading to the appearance of the hourglass pattern is that the DGEN should gradually get sparser as development progresses, with general-purpose regulatory genes at the earlier developmental stages and highly specialized regulatory genes at the later stages, expressing the oldest genes during mid development. We suggest to also include findings investigating the functionality and age of the genes that have been shown to be active during mid embryogenesis to correlate potential outcomes of the DGEN prediction that evolutionary older genes should be concentrated at the waist of the hourglass with results obtained by recent studies 3 4 5 6 7 8 9 10 11 12. These aforementioned studies were able to show that during mid embryogenesis evolutionary old genes or evolutionary old processes that are shared within and between phyla are most active. In our opinion it would be very interesting to test the degree of intersection between genes predicted to be causal for the waist of the DGEN hourglass and evolutionary conserved genes that have been found to be expressed during mid embryogenesis. We are aware that the model uses the number of genes at each developmental stage combined with the age of the corresponding genes to observe constraints during development. Hence, only the authors can estimate how much effort would be needed to identify such intersecting genes. If the search for intersecting genes returned by the DGEN model during mid development and genes found by previous studies should be possible, the authors could integrate a motivation section to find these intersecting genes within the Introduction section.”Summary: “Correlate the output of the DGEN simulation with existing findings of studies addressing similar questions of the causality of the developmental hourglass phenomenon and discuss potential simplifications of the model assumptions that do not fit experimental results or that are not derivable via experiments yet.”Response: This is a very interesting idea and we thank the reviewers for this suggestion. We are planning to pursue this investigation in a follow-up paper that will also include the previously mentioned analysis of sea-urchin GRNs as well as the DSI/TSI results. We believe that it would be distracting to try to include all these results in this first paper, given that the main focus of this work has been on the computational model and the computational results. We are also open to collaborate in this investigation with the reviewers or other researchers working in this area, if they are interested. Review: “The model description is quite intuitive. However, as the validity of the whole study depends on the model parameters chosen, several points need clarification including the rationale and motivation to choose exactly these parameters. One of the most crucial parameters of the model is the regulatory specificity s(l), with 0 <= s(l) <= 1. We think, that adding a more detailed paragraph discussing how s(l) is being estimated, would be highly beneficial due to the importance for the parameter s(l) for the entire model and predictions. In Model-1-4 the authors choose three different values for s(l): In Model-1: s(l) = 0.5 and in Model-2-4: s(l) = l/L, and in Figure 9 a non-linear function s(l) = 0.9 – (0.8 / 1 + exp(\\gamma - l) ). A gene g at stage l is modeled to act as upstream regulator for a gene g’ at stage l + 1 with probability s’(l) = 1 – s(l). From this follows that each potential upstream regulator in stage l has the same probability s’(l) to regulate genes g’ in stage l + 1. In other words, Model-1 assigns each potential upstream regulator with the same probability s’(l) = 1 – 0.5 = 0.5 to regulate genes g’ in stage l + 1, in Model-2: s’(l) = 1 – l/L = const., and for the non-linear function s(l): s’(l) = 1 – (0.9 – (0.8 / 1 + exp(\\gamma - l) )) = const. It would be advantageous, if the authors could include motivations for the three cut-offs, especially how they derived the sigmoid-like mathematical function (Figure 10, legend caption) they later denote as non-linear function s(l). We do see that modeling the stage specificity with a constant value, an increasing value, and a non-linear function is plausible, but a clear motivation or derivation of the exact values would be beneficial for a better understanding of the modeling process.The authors discuss that their major assumption is that the regulatory specificity increases substantially as development progresses, hence the DGEN becomes gradually sparser along the developmental time axis starting with s(1) ~ 0, … , s(L) ~ 1. Here the authors should include references to experimental observations that support their assumption. Since Models 2-4 rely on this assumption, published experimental studies in line with this assumption need to be referenced. Otherwise, the models applied would seem largely academic.Include a clear statement how the exact values of the stage specificity (regulatory specificity) s(l) have been derived. State more clearly that s(l) is the crucial parameter determining the observed phenomena.”Response: The regulatory specificity functions we experimented with are arguably the simplest one could think of. Specifically, we start (Model-1) with the simplest hypothesis that the regulatory specificity does not vary across developmental stages (in the paper we show results for s=0.5 but we have generated results for all values 0.1, 0.2, ... 0.9 and the conclusion remains that when the specificity is constant the network does not evolve into an hourglass shape). Then, we consider (Model-2) a linearly increasing specificity function. We do not argue that this is realistic or that there are experimental results that suggest this linearity or even the increasing trend. It is just the simplest and most parsimonious model that leads to the emergence of an hourglass pattern. Finally, in Figures-9 and 10 we consider a more general specificity function that increases in a non-linear manner, providing a simple way to control the stage at which it gives the mid-range value 0.5. This allows us to examine how the shape of the specificity function s(l) affects the location of the hourglass waist. We hope that follow-up work will reveal experimentally the actual shape of the regulatory specificity function s(l) and there is no doubt that it will not be identical to any of these simple mathematical functions.Please note that we have revised the paragraphs about “Model-1” and “Model-2” in the Simulation section to clarify these points. Review: “For the probabilities P_{DL} and P_{DP} the explicit formula to compute the corresponding probabilities could also be included within the paragraph analogous to P_{RF}. This would enable a clearer reproducibility.“Response: Actually there is no formula for the gene deletion and duplication probabilities. They are given probability values that do not depend on any other parameters. At each round of the simulation, a gene is duplicated with probability P_{DP}. If not rewired or duplicated, the gene is deleted with probability P_{DL}. Review: “Hourglass score H: Here w(l) is defined as the number of transitioning genes in stage l. In Figure 3 and in section 'Hourglass shape' w(l) is referred to as stage width. In case the definition of w(l) is equivalent to the stage width, it would be beneficial for the reader to see the term stage width in the same sentence as the initial definition of w(l). A correct understanding of the stage width is important to understand the measurement of H.”Response: The reviewer is right. We will use the “stage width” term when we first introduce the notation w(l). Review: “Furthermore, the univariate Mann-Kendall statistic should be referenced to allow a non-statistical community to better understand this way of statistical modeling.”Response: Please see how we addressed a similar comment (comment #5) in Dmitri Krioukov's review. Review: “Transcriptome age index (TAI): In this section the authors describe and perform two different methods to assign each gene a corresponding evolutionary age. For D. melanogaster they collected groups of orthologs from OrthoDB and OrthoMCL, whereas for A. thaliana they obtained phylostratum assignments from Quint et al.10 that was derived by phylostratigraphy[ref-13.] We do not understand the motivation of merging orthologous gene groups obtained from OrthoDB and OrthoMCL, as both approaches apply different parameters and heuristics to determine orthologous genes. Was this done to maximize the number of potential orthologs to be included in the study? In addition, why did the authors use a different approach for the plant gene set? This seems somewhat inconsistent and statement concerning the plausibility to compare results returned by the DGEN model based on age assignments obtained from phylostratigraphy (A. thaliana) with OrthoDB and OrthoMCL merged orthologs (D. melanogaster) would be appreciated. Furthermore, the database specific parameters they used in OrthoDB and OrthoMCL should be mentioned as well as the database version they used, e.g. OrthoDB2, or OrthoDB3, … , or OrthoDB6.”Summary: “Explain why merging OrthoDB results with OrthoMCL results might be plausible and why comparing it with results obtained from phylostratigraphy is reliable.”Response: The phylostratigraphy technique as employed by Domazet-Loso et al. and Quint et al. involves the assignment of an evolutionary age to each gene in a given species, by tracing the most distant ancestral node containing at least one immediate daughter species with a detectable homologue. A phylogenetic tree of divergent species ranging from cellular organisms transitioning into simple and more complex eukaryota served as a reference to divide phyla radiating from consecutive ancestral points into distinct phylostratum layers. To determine the homologues, BLAST sequence similarity searches were done against complete genomes reliably annotated across the different phylostrata. Similarly, we obtained known homologues of each Drosophila gene from OrthoDB5 and OrthoMCL5 (that also use BLAST based searches) in the species depicted in Figure 4, and mapped them to six different “phylostrata” (marked in Figure 4). Akin to the method described in the above references, we assigned each gene to a specific “phylostratum” based on the farthest detectable homologue of that gene, and this is referred to as “age index” in the paper. This made it possible to compare the age index based results we obtain in Drosophila to the phylostratigraphy based assignments in Arabidopsis.Homologues for Drosophila genes were sourced from these two databases, to increase coverage across the species depicted in the tree in Figure 4. Since these two databases use slightly different parameters to identify orthologs, we examined whether the usage of specific databases introduce bias by analyzing data from each database separately and observed that age index of each gene remained the same. Review: “Age index for each stage-pair: There seems to be a typo within the TAI formula. As defined by Domazet-Loso and Tautz6 and adapted to the author’s notation the formula of the TAI should begin TAI(l) not TAI(1).”Response: We have fixed this problem. Thank you. Review: “The authors show based on their findings returned by Model-2-4 that the increasing specificity assumption is the key property behind the developmental hourglass effect. We think that this statement is not clear enough. The authors could phrase more clearly what they mean by “key property behind the hourglass effect” inferred from Model-2-4.”Response: The reviewer is right. We revised that paragraph as follows:“In Model-1 and Model-2, genes can be only removed (due to RW events, potentially followed by RF cascades) and so the average DGEN size decreases as evolutionary time progresses, which is unrealistic. Model-3 and Model-4 are more realistic because they can maintain a roughly constant DGEN size in the long-term. However, as will be shown next, all aspects of the developmental hourglass effect can already be seen with Model-2 (but not with Model-1). This highlights that the increasing specificity assumption is sufficient to generate the hourglass effect. Further, the inclusion of additional biological mechanisms in the model, namely gene duplication and gene deletion, even though they make the model more realistic, they are not necessary for the emergence of the hourglass effect.\" Review: “Age of genes: Here the authors motivate that defining the age of a gene as A(g) = i – t0(g) is plausible, because a rewiring event may give a gene a new function, at least in terms of its upstream and downstream regulators. This statement needs validation from the literature and from experimental studies that already tested this hypothesis for specific rewiring events.”Response: We have added two references that provide some evidence to the previous hypothesis: Guet et al. (2002) and Kim et al.(2012). Review: “Furthermore, the authors show (Figure 8c) that the evolutionary age at stage l follows the same pattern as the lethality probability: gradually increasing until the waist of the hourglass, and then gradually decreasing. This finding should be correlated with the findings by Galis and Metz3 and should be discussed more clearly in the Discussion.“Response: The reviewer is right. The findings of Galis and Metz are highly relevant and consistent with our computational results regarding the lethality probability. We modified the paper as follows:In the Results section:“These computational results for the lethality probability across development are consistent with the empirical observations of Galis and Metz \\cite{galis2001testing} about the increased mortality of rodents due to perturbations in the phylotypic stage (see Discussion section).\"In the Discussion section:“The third prediction of the model is in direct agreement with the empirical observations of Galis and Metz regarding the increased mortality caused by perturbations during the phylotypic stage \\cite{galis2001testing}. That study has shown, based on the teratological literature for rodent development, that disturbances in the phylotypic stage lead to much higher mortality than in other stages. Further, such disturbances lead to the co-occurrence of several distinct anomalies in the developing embryo and so the increased mortality cannot be due to a single particularly vulnerable process that takes place at that stage.” Review: “In the first sentence of the last paragraph before the Discussion, \"… and TAI profiles from Arabidopsis\" should be referenced to reference 11 and not 12.“Response: Thank you, we have fixed this problem.Review: “Discussion: The authors discuss that the observed pattern of conservation (developmental hourglass) may stem from fundamental organization principles and that the exact origin of these principles remains elusive. Here the authors should include existing studies that also aim at predicting a universal organization principle during development (modularization, etc.) and what exact problems are preventing studies to elucidate this universal phenomenon. We do note, that the authors cite studies that aim to explain the effects causing the early phase of the developmental hourglass 14 15 16 and studies aiming to explain effects causing the late phase of the developmental hourglass1, but we think that it should be explicitly stated that the model is designed to test aspects of Raff’s initial hypothesis and that also more recent studies have been investigating the underlying reasons for the pattern in the early phase of the hourglass17.”Response: The reviewer is right. We have revised the Discussion section to also include a paragraph about Raff’s hypothesis (see our response to comment 1). We have also added another paragraph (see below) to discuss a very recent paper that proposes a different mechanism for the emergence of the hourglass effect.“Recently, Friedlander et al. have proposed a different mechanism for the emergence of hourglass-like patterns in evolving biological or technological networks, based on a linear system formulation \\cite{friedlander2014evolution}. They showed that if a system can be represented as a hierarchical and layered linear transformation of an input vector to an output vector, and the desired transformation matrix is rank-deficient, then an evolutionary process that selects that particular transformation can, under certain conditions, converge to an hourglass-like structure. It is not clear yet how to adapt this linear model in the context of inherently non-linear systems, such as gene regulatory networks.”" } ] }, { "id": "5650", "date": "31 Jul 2014", "name": "Dmitri Krioukov", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article proposes and analyzes a network model that is capable of reproducing the hourglass effect in the number of genes that undergo a functional state transition as a function of the developmental stage. The hourglass effect refers to higher morphological divergence at earlier or later stages of embryonic developmental, compared to medium stages. The main idea of the model is to explain the waist of this hourglass (the number of \"transitioning genes\" w(l) having a minimum at medium developmental stages l) by an interplay between the impact of random rewiring events in the developmental gene execution network (DGEN) and stage specificity. The DGEN is a network representing regulatory interactions between genes at different developmental stages, while stage specificity s(l) defines the probability 1-s(l) with which a gene at stage l regulates a gene at stage l+1. One of the key assumptions in the model is that s(l) is an increasing function of l. As a result of the interplay between a decreasing impact of DGEN perturbations as a function of l, and increasing s(l), the lethality of perturbations is maximized at mid l's, leading to evolutionary incentives to minimize w(l) at those mid stages. The article consists of three parts. The first part describes the model in detail. The second part discusses extensive simulation results of the model. The third part presents an extensive analysis of existing developmental data on Drosophila melanogaster and Arabidopsis thaliana in the model context.The most interesting aspect of the article is that provides a possible intriguing explanation of the hourglass effect in developmental biology, which may foster future creative thinking and research in this important direction. The most obvious reservation one can express about the article is that the model cannot be currently refuted since it is formulated at the DGEN level, and there is currently no data from which a reliable DGEN reconstruction would be possible. The fact that some outcomes of model simulations qualitatively agree with available data does not directly validate either the model, or its main assumptions. The article does not contain such claims, however, that would not be supported by the data.My minor comments that may help to improve the article are:The main idea of the model is buried somewhere on page 6 in the middle of a paragraph after sentence \"What is the reason behind the hourglass shape of DGENs?\" Why not explain this as early as possible, certainly in the Introduction, if not in the abstract? The third paragraph in the Introduction reads somewhat unclear and imprecise, as well as the \"Developmental gene execution networks\" section. When D(g) and Gamma notations first appear, it's not spelled out what they are. DGEN removals upon DF events have a clear meaning. But what do replacements correspond to in reality? The Mann-Kendall statistics could be briefly described, or at least a reference could be provided.", "responses": [ { "c_id": "1124", "date": "18 Dec 2014", "name": "Constantine Dovrolis", "role": "Author Response", "response": "First, we would like to thank the author of this review for his thorough reading of our work. His comments have been very helpful in revising this paper (please see Version 2). We would also like to apologize for not revising the paper earlier.Review: “The most obvious reservation one can express about the article is that the model cannot be currently refuted since it is formulated at the DGEN level, and there is currently no data from which a reliable DGEN reconstruction would be possible. The fact that some outcomes of model simulations qualitatively agree with available data does not directly validate either the model, or its main assumptions. The article does not contain such claims, however, that would not be supported by the data.”Response: It is true that a direct validation of the model is not possible with any currently available data (at least to the extent of our knowledge). Such a direct validation would require the reconstruction of the complete DGEN for specific model organisms, i.e., knowing not only the expression profile of each gene as a function of developmental time and at different tissues of the embryo but also knowing the regulatory inputs for each gene during development. However, there are research groups that are working in that direction (e.g., Prof. Davidson’s group at Caltech is gradually “reverse-engineering” the DGEN of the sea-urchin). Our hope is that a direct validation of our model will be possible within the next few years.Having said that however, even though our model cannot be directly validated, we are providing indirect evidence that certain predictions of the model are true in Drosophila and Arabidopsis (see Results - Data Analysis section). Consequently, the model should not be viewed as completely hypothetical either. Review: “The main idea of the model is buried somewhere on page 6 in the middle of a paragraph after sentence \"What is the reason behind the hourglass shape of DGENs?\" Why not explain this as early as possible, certainly in the Introduction, if not in the abstract?”Response: The reviewer is right. We revised the fourth paragraph of the introduction to include a “hint” about the main reason behind the hourglass effect. Obviously, we cannot write much more at that early point of the paper because we still haven’t defined what we mean by DGEN, RFs, cascades, etc. The revised paragraph is:“The model predicts that the evolutionary process shapes the DGENs of a population in the form of an hourglass, under fairly general assumptions. Specifically, the number of genes at each developmental stage follows an hourglass pattern, with the smallest number at the “waist” of the hourglass. The main condition for the appearance of the hourglass pattern is that the DGEN should gradually get sparser as development progresses, with general-purpose regulatory genes at the earlier developmental stages and highly specialized regulatory genes at the later stages. Under this assumption, the model predicts that gene regulatory changes or rewiring in mid-development are more likely to cause cascades of removing non-essential genes from the DGEN, compared to early or late developmental stages. Another model prediction is that the evolutionary age of DGEN genes also follows an hourglass pattern, with the oldest genes concentrated at the waist.” Review: “When D(g) and Gamma notations first appear, it's not spelled out what they are.”Response: We checked this. The notation D(g) is introduced at the end of the “Developmental gene execution networks” section, and that is the first time it appears in the paper. The notation Gamma first appears in the “Developmental Failure (DF)” paragraph, and that is also where it is defined. Review: “DGEN removals upon DF events have a clear meaning. But what do replacements correspond to in reality?”Response: DGEN replacements model the effect of selection: an embryo that did not develop properly (DF) dies and its DGEN is removed from the population, while healthy embryos develop and eventually give birth to new a embryo with the same genotype as its parent (asexual reproduction). Review: “The Mann-Kendall statistics could be briefly described, or at least a reference could be provided.”Response: We have added a reference for this statistic:\"Gibbons JD, Chakraborti S: Nonparametric Statistical Inference. Marcel Dekker New York. 2003. Reference Source”" } ] }, { "id": "5391", "date": "06 Aug 2014", "name": "Gourab Ghoshal", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Akhshabi et al. proposes and describes in detail an evolutionary model that qualitatively reproduces the \"Hourglass Effect\"---a phenomenon (in the biological context) whereby there is increased morphological divergence at early and late stages of embryonic development separated by increased conservation in the phylotypic stage. The proposed model is based on a hierarchical network representation of the Gene Regulatory Network - the so-called Developmental Gene Execution Network (DGEN) - and evolution proceeds through a series of stochastic perturbations involving gene duplication, re-wiring and deletion. The manuscript suggests that the key factor that reproduces the hourglass effect is the monotonicity in \"time\" (increasing) of a function they call specificity s(i) (a measure of likelihood of a gene regulating descendant genes at later times) that competes against perturbative effects such that there is a \"waist\" of the number of transitions genes w(l) at intermediate times. They then test predictions of the model in two datasets of developmental data Drosophilia melanogaster and Arabidopsis Thaliana, finding good qualitative agreement. The manuscript is properly motivated and well written (with some caveats, see comments below). The model proposed is quite intuitive and compelling and represents an excellent first attempt in beginning to uncover the mechanism behind this intriguing phenomenon. I anticipate that this will have high impact (considering the hourglass effect transcends biological phenomena and is also found in \"designed\" systems such as the Internet protocol stack) in multiple fields. Consequently I strongly endorse this manuscript. I do however have some concerns about some of the details about the model and therefore propose the following that may improve the readability of the manuscript and the plausibility and robustness of the model:The section describing the model is a bit dense, and can benefit from some rewriting. For example it is a bit hard to follow what the significance of a spatial domain is and its relevance to the model. In addition a bewildering plethora of parameters are introduced, that can be a bit overwhelming for the reader. Consequently I suggest putting all the important and relevant parameters in a table for easy access to the reader. The section on Rewiring (RW) is quite hard to read and will benefit from the introduction of explicit equations similar to that for P_{RF}. Which brings me to my second point about the assumptions of the model. It seems to me that two key factors lead to the hourglass effect: a) the montonocity of s(l) with stage l and likewise b) of P_{RF} with r. While the choice of this dependence for the specificity is a simple linear dependence (which makes sense as a first pass), the choice of function for P_{RF} is highly non-trivial. Why not for example choose a simple linear dependence such as P_{RF} ~ r? The reason why I say this is that the authors would like to propose the most general and minimal model to explain this phenomenon. And it is not clear to me whether the specific choice of the functional dependences matter. For example with a non-linear choice of P_{RF} we see that a non-linear s(l) has the effect of shifting the waist. But what about a linear choice of P_{RF} and s(l)? Does that still preserve the hourglass effect? In my opinion a truly robust model will state that the main things that determine the hourglass are the monotonic dependencies in combination with non-linear or linear choices of the functions independent of their specific forms. This will allow the model to be applied to multiple settings.At the moment I'm a bit concerned that the non-trivial choice of P_{RF}, as it stands in the manuscript, might be key to the observed effect. If it is indeed so, then one must motivate why one must make this particular choice. Same goes for the slightly peculiar choice for the non-linearity of s(l) introduced later in the model. The authors will do well to explain in detail what motivated them to make such a choice. Additionally it would be helpful if the authors make it clearer (than they already have) that effects such as duplication and deletion are there only to make the model more realistic in terms of maintaining the number density of genes across different stages and play no part in the hourglass effect (or at least that's how I understand it). Some minor nitpicks:In the section Model Description, the authors refer to the Wright-Fisher model. I was not aware of what this was and had to look it up, so a reference for the reader should be provided. The Hourglass score H is determined through Mann-Kendall statistics. A brief description of the method should be provided for the benefit of the reader (maybe in the supplementary material). Additionally, why this particular choice? Presumably any statistical test for monotonic trends should be robust to the parameters of the model. Would there be much of a difference is one used the Spearman rho for example? Finally I think the authors will do well to highlight the importance of Specificity right at the outset of the manuscript and move some simplified variant of the segment \"What is the reason behind the hourglass shape of DGENS?\" (page 7, line 11, second column) to somewhere in the introduction.", "responses": [ { "c_id": "1123", "date": "18 Dec 2014", "name": "Constantine Dovrolis", "role": "Author Response", "response": "First, we would like to thank the author of this review for his very thorough reading of our paper. His comments and suggestions have been extremely helpful in revising the paper (please look at Version-2). We would also like to apologize for not revising the paper earlier.Review: “The section describing the model is a bit dense, and can benefit from some rewriting. For example it is a bit hard to follow what the significance of a spatial domain is and its relevance to the model.”Response: We hope our extensive revision has mitigated this problem of the previous version of the paper.We explain what we mean by “spatial domains” early in the paper (and in Fig-1) because it is an important and necessary concept when we justify later in the paper the assumption of increasing regulatory specificity (see paragraph that starts with “The major assumption is that the regulatory specificity…”). It is the formation of those distinct spatial domains (or “ modules”) that explains why the regulatory specificity is probably decreasing as development progresses. Review: “The section on Rewiring (RW) is quite hard to read and will benefit from the introduction of explicit equations similar to that for P_{RF}.\"Response: We have revised that paragraph to make it more clear. Review: It seems to me that two key factors lead to the hourglass effect: a) the monotonicity of s(l) with stage l and likewise b) of P_{RF} with r. While the choice of this dependence for the specificity is a simple linear dependence (which makes sense as a first pass), the choice of function for P_{RF} is highly non-trivial. Why not for example choose a simple linear dependence such as P_{RF} ~ r? For example with a non-linear choice of P_{RF} we see that a non-linear s(l) has the effect of shifting the waist. But what about a linear choice of P_{RF} and s(l)? Does that still preserve the hourglass effect? In my opinion a truly robust model will state that the main things that determine the hourglass are the monotonic dependencies in combination with non-linear or linear choices of the functions independent of their specific forms.”Response: We chose the non-linear P_{RF} function shown in Fig-2 because it is quite flexible and it allows us to examine how the shape of this curve affects the behavior of the model by simply varying the parameter z. Please note that when z is equal to one this function is almost linear. As discussed in Fig-10 however, as z decreases towards one the location of the hourglass waist also decreases. Consequently, we expect that a linearly increasing P_{RF} function would result in a shape that resembles a “funnel” rather than an hourglass.We hope that new experimental work will provide some direct evidence for the shape of this function in the near future.Review: “In the section Model Description, the authors refer to the Wright-Fisher model. I was not aware of what this was and had to look it up, so a reference for the reader should be provided.” Response: We have added a reference for this model:“Ewens WJ. Mathematical Population Genetics. Springer-Verlag Berlin New York. 1979. Reference Source” Review: “The Hourglass score H is determined through Mann-Kendall statistics. A brief description of the method should be provided for the benefit of the reader (maybe in the supplementary material). Additionally, why this particular choice? Presumably any statistical test for monotonic trends should be robust to the parameters of the model. Would there be much of a difference is one used the Spearman rho for example?”Response: We have added a reference for this statistic:\"Gibbons JD, Chakraborti S: Nonparametric Statistical Inference. Marcel Dekker New York. 2003. Reference Source”The Mann-Kendall statistical test is one of the most widely used statistics to assess the significance of trends in data (Gibbons and Chakraborti 2003). Mann-Kendall and Spearman’s tests perform highly similar in data analyses (e.g., Yue et al., 2002; Shadmani et al., 2012). Review: “Finally I think the authors will do well to highlight the importance of Specificity right at the outset of the manuscript and move some simplified variant of the segment \"What is the reason behind the hourglass shape of DGENS?\" (page 7, line 11, second column) to somewhere in the introduction.”Response: The reviewer is right. We revised the fourth paragraph of the introduction to include a “hint” about the main reason behind the hourglass effect. Obviously, we cannot write much more at that early point of the paper because we still haven’t defined what we mean by DGEN, RFs, cascades, etc." } ] } ]	1	https://f1000research.com/articles/3-156
https://f1000research.com/articles/3-277/v1	14 Nov 14	{ "type": "Research Note", "title": "A common feature pharmacophore for FDA-approved drugs inhibiting the Ebola virus", "authors": [ "Sean Ekins", "Joel S. Freundlich", "Megan Coffee", "Joel S. Freundlich", "Megan Coffee" ], "abstract": "We are currently faced with a global infectious disease crisis which has been anticipated for decades. While many promising biotherapeutics are being tested, the search for a small molecule has yet to deliver an approved drug or therapeutic for the Ebola or similar filoviruses that cause haemorrhagic fever. Two recent high throughput screens published in 2013 did however identify several hits that progressed to animal studies that are FDA approved drugs used for other indications. The current computational analysis uses these molecules from two different structural classes to construct a common features pharmacophore. This ligand-based pharmacophore implicates a possible common target or mechanism that could be further explored. A recent structure based design project yielded nine co-crystal structures of pyrrolidinone inhibitors bound to the viral protein 35 (VP35). When receptor-ligand pharmacophores based on the analogs of these molecules and the protein structures were constructed, the molecular features partially overlapped with the common features of solely ligand-based pharmacophore models based on FDA approved drugs. These previously identified FDA approved drugs with activity against Ebola were therefore docked into this protein. The antimalarials chloroquine and amodiaquine docked favorably in VP35. We propose that these drugs identified to date as inhibitors of the Ebola virus may be targeting VP35. These computational models may provide preliminary insights into the molecular features that are responsible for their activity against Ebola virus in vitro and in vivo and we propose that this hypothesis could be readily tested.", "keywords": [ "ebola virus", "computational models", "machine learning" ], "content": "Introduction\n\nThe current Ebola virus (EBOV) crisis has demonstrated that globally we are not prepared to respond with therapeutics to treat existing infections or act as prophylactics as there is no Food and Drug Administration (FDA) or European Medicines Agency (EMEA) approved therapeutic. More importantly this suggests we should have been prepared for a pathogen which has been known about for nearly forty years. The current EBOV outbreak is already proving remarkably costly in terms of the mortality and financial ramifications1,2. The best approaches to EBOV so far have relied on public health measures for containment3 which have been used in past outbreaks4. These lessons with EBOV will undoubtedly be important for the next virus outbreak5 but they also raise many questions6 which point to how little we know about these viruses in general, as well as how best to share knowledge openly7.\n\nThere have been a relatively small number of studies that have attempted to identify compounds active against EBOV. Two recent studies utilized high-throughput screens of a subset of FDA approved drugs against different EBOV strains (Zaire and Sudan) in vitro and in vivo. These independent reports suggested the promise of the antimalarials amodiaquine and chloroquine in one study8, while the selective estrogen receptor modulators (SERMs) clomiphene and toremifene were active in another9. Chloroquine to date has not progressed beyond the mouse EBOV model used in these studies. We hypothesized that we could use these four molecules to computationally define the features that are important for activity. The previous studies were not exhaustive screens of all FDA drugs and so we have taken this opportunity to suggest additional compounds. Looked at from another perspective “non-antiviral” drugs may be worth following up even though their molecular mechanism is unknown. These compounds may themselves have broad antiviral activity as reports describe modest inhibitory activity against other viruses10–13.\n\nSeveral studies have identified non-FDA approved drugs including an in silico docking approach to identify molecules targeting the viral Nedd4-PPxY interface14. These molecules were similar to the FDA benzimidazole and aminoquinoline8,9 compounds that were active against EBOV. Another good example is the recent in silico docking of 5.4 million drug-like compounds docked in the viral protein VP35 protein15. This identified multiple pyrrolidinones which inhibit its polymerase cofactor activity15. The pyrrolidinones bind to an alpha helix which is proposed as important for viral function16. With the limited knowledge of small molecules and potential targets we have studied whether the FDA-approved drugs that are active in vitro and in vivo versus EBOV could be targeting VP35.\n\n\nMethods\n\nTwo papers from 2013 described compounds active as inhibitors of different EBOV strains in vitro and in vivo, namely amodiaquine and chloroquine in one study8, clomiphene and toremifene in another9. These active molecules were used as they have both in vitro and in vivo activity to build a common features pharmacophore with Discovery Studio 4.1 (Biovia, San Diego, CA) from 3D conformations of the molecules generated with the CAESAR algorithm. This identified key features. The pharmacophore was then used to search various databases (for which up to 100 molecule conformations with the FAST conformer generation method with the maximum energy threshold of 20 kcal/mol, were created). The pharmacophore was then used to search the Microsource Spectrum database (http://www.msdiscovery.com/spectrum.html) as well as the CDD FDA drugs dataset (https://www.collaborativedrug.com/pages/public_access). In both cases over 300 hits were retrieved initially. The van der Waals surface of amodiaquine (which was more potent than chloroquine8) was added to limit the number of hits retrieved17–19.\n\nReceptor-ligand pharmacophores for the VP35 protein were generated from crystal structures (4IBB, 4IBC, 4IBD, 4IBE, 4IBF, 4IBG, 4IBI, 4IBJ, 4IBK) in the protein data bank PDB. Pharmacophores were constructed using the receptor-ligand pharmacophore generation protocol in Discovery Studio version 4.1 (Biovia, San Diego, CA) with a maximum number of pharmacophores (10), minimum features (4), and maximum number of features (6) as are described elsewhere20.\n\nPDB 4IBI was used for docking using LibDock in Discovery Studio (Biovia, San Diego CA)21. The proposed binding site was centered on the ligand and a site sphere created (coordinates 2.14, 20.93, 1.71) with 9.45 Å diameter. The protocol included 10 hotspots and docking tolerance (0.25). The FAST conformation method was also used along with steepest descent minimization with CHARMm. Further parameters followed the default settings. The ligand VPL57 was removed from the binding site and re-docked. The four FDA approved drugs with activity against Ebola were docked in the structure from an sdf file. Molecules were visualized alongside the original ligand VPL57 and the 2D interaction plots generated.\n\n\nResults\n\nThe pharmacophore was generated using the in vivo and in vitro active amodiaquine, chloroquine, clomiphene and toremifene (Supplemental Table 1) as these represent the most relevant FDA approved drugs to date. This pharmacophore consists of 4 hydrophobic features and a hydrogen bond acceptor feature (Figure 1). The pharmacophore with van der Waals surface was also used to search FDA drug various libraries (Supplemental Table 2 and Supplemental Table 3). The most interesting observations from this virtual screen are that various estradiol analogs score well (e.g. estradiol valerate Fit value 4.23). Previously estradiol was suggested to be active in the EBOV pseudotype assay in vitro8. In addition, dibucaine was also retrieved (Fit value 1.58) which was also active in the EBOV pseudotype assay8. Amodiaquine, chloroquine, clomiphene and toremifene can be used as positive controls for future screens. Because the original complete sets of FDA approved compounds screened are not publically accessible it is difficult to compare hit rates versus all compounds tested to date.\n\nA. amodiaquine, B. chloroquine, C. clomiphene D. toremifene and E. Overlap showing all molecules in the van der Waals surface of amodiaquine.\n\nThe nine receptor-ligand pharmacophores created all consisted of three to four hydrophobic features and one to two hydrogen bonding features (Table 1). Eight of these pharmacophores also had a negative ionizable feature. These suggest that the receptor-ligand based approach results in a general similarity across the nine structures, likely indicating the similar binding mode and importance of features for interfering with this generally hydrophobic pocket for protein-protein interactions.\n\nPharmacophores were generated using the receptor-ligand pharmacophore generation protocol in Discovery Studio version 4.1 (Biovia, San Diego, CA) with minimum features (3) and maximum features (6). Pharmacophore features are Hydrophobic (H, cyan), Hydrogen bond acceptor (HBA, green), hydrogen bond donor (HBD, purple) and 1 negative ionizable (neg, blue). Excluded volumes (grey) were also automatically added. Further details on this approach are described elsewhere20.\n\nRedocking the 4IBI ligand in the protein resulted in an RMSD of 3.02Å, which generally indicates the difficulty of predicting orientations for compounds binding in what is a relatively hydrophobic and shallow pocket (Figure S1). This molecule was ranked the 29th pose and had a LibDock score of 86.62 (Figure S1). The four FDA approved drugs were docked into the VP35 structure 4IBI. All compounds docked similarly and overlapped with the co-crystal ligand (Figure 2). Amodiaquine and chloroquine had higher LibDock scores (> 90) than the 4IBI ligand, while clomiphene and toremifene had LibDock scores less than 70. All four FDA approved drugs bound similarly to the pyrrolidinone ligands in the pocket formed by residues from the α-helical and β-sheet subdomains15. We have highlighted proposed energetically favorable interactions of the antimalarial candidate binders with ILE295, LYS248 and GLN244, which scored favorably. Previously published studies suggested mutation of ILE295, LYS248 resulted in near-complete loss of binding activity15.\n\n4IBI was used, 4IBI ligand VPL57 shown in yellow. A. Amodiaquine (grey) and 4IBI LibDock score 90.80, B. Chloroquine (grey) LibDock score 97.82, C. Clomiphene (grey) and 4IBI LibDock score 69.77, D. Toremifene (grey) and 4IBI LibDock score 68.11\n\n\nDiscussion\n\nOur previous experience with common feature and quantitative pharmacophore models has demonstrated their value in predicting novel actives from collections of FDA approved drugs22–27. Candidate predicted actives may be assessed by their Fit Value to the pharmacophore model. This score can be used to prioritize compounds for eventual testing. In the current study it was hypothesized that two different classes of compounds showing activity against EBOV in vitro and in vivo may share a common pharmacophore. Construction of this pharmacophore (Figure 1) indicated four hydrophobic features and a hydrogen bond acceptor feature. This pharmacophore (with an added van der Waals surface to limit the number of hits retrieved) was then used to screen and score other FDA drugs from a small database and identified 120 and 124 structures for future evaluation in vitro testing (Supplemental Table 2 and Supplemental Table 3). Out of these compounds estradiol and dibucaine had been previously described as active in in vitro EBOV assays. This suggested the pharmacophore could retrieve some structurally diverse classes of known hits8.\n\nRecently identified co-crystal structures of the EBOV VP35 protein were used to derive receptor-ligand pharmacophores. These nine receptor-ligand pharmacophores suggested the importance of hydrophobic, hydrogen bonding and negative ionizable interactions to interfere with this protein-protein interaction (Table 1). Eight out of nine of the pharmacophores had one or more hydrogen bond acceptor feature. These pharmacophores are grossly similar to our ligand based pharmacophore (derived from four FDA approved drugs that inhibit EBOV), as both types of model had multiple hydrophobic features and at least one hydrogen bond acceptor. When we docked the antimalarials and SERMs into a representative VP35 structure these compounds were found to overlap with the X-ray ligand to differing extents. Amodiaquine and chloroquine had LibDock scores greater than 90 and higher than that for the redocked X-ray ligand. This indicated that VP35 may be a potential target for these two distinct classes of compounds. However, it is important to point out that we have not compared docking to other proteins in EBOV and it could also be possible that these molecules are active elsewhere as well as via other mechanisms than by specific binding to proteins28,29. Further, VP35 may be a preferred target for the antimalarials while the SERMs are not predicted to bind as well as the X-ray ligand. The use of other docking and scoring methods may produce differences in the pose and predicted binding affinity, which could be of interest for further studies.\n\nA combination of the promising efficacy of chloroquine (EC50 16 μM8) and amodiaquine (EC50 8.4 μM8) versus EBOV, their availability and likely low cost should prioritize their further laboratory exploration. Mechanistic studies against VP35 and possibly other proteins should also be pursued and may be enlightened by the observation that both of these compounds also have reported activity against other viruses. For example, chloroquine is active against human coronavirus OC43 (in vitro and in infected mice) as well as SARS (in vitro)10,30,31, while amodiaquine also inhibits dengue virus 2 replication and infectivity in vitro11.\n\n\nConclusions\n\nIn summary, this study has built on the previous publications that identified four FDA approved compounds active against different strains of EBOV8,9. Our pharmacophore model for SERMs and aminoquinolines suggests that these compounds share multiple chemical features based on their overlap to the four hydrophobic features and a hydrogen bond acceptor (Figure 1E) and they may have a common mechanism or target. We suggest that VP35 may be the likely target based on the overlap of receptor-based pharmacophores and docking into the crystal structure. Amodiaquine and chloroquine score particularly well in terms of docking to VP35. If this is the case it could provide a means to follow up with other small molecule analogs and/or additional FDA approved drugs that could target this protein-protein interaction. As with our other tuberculosis-focused research32,33, and computational approaches to repositioning compounds34 we embrace the essentiality for computational predictions to be interrogated through rigorous experimental studies. For example at least two in silico docking studies screened commercially available compounds14,15. We propose that docking FDA approved drugs could also be a viable first step to identifying potential compounds that could be used. We are actively seeking collaborators with experience with EBOV assays to enable further translational studies. We believe this computationally inspired approach may be applicable for other known infectious pathogens that do not have current treatments such as other viruses related to Ebola. Ultimately we need to be able to leverage such approaches to provide antivirals for future pathogens.\n\n\nData availability\n\nF1000Research: Dataset 1. Pharmacophores, receptor ligand models and docking data for FDA-approved drugs inhibiting the Ebola virus, 10.5256/f1000research.5741.d3844935.\n\nThe ligand-based pharmacophore was previously made available: http://figshare.com/articles/Ebola_active_cpds_pharmacophore/1190902.\n\nThe following PDB structures were used in this study (4IBB, 4IBC, 4IBD, 4IBE, 4IBF, 4IBG, 4IBI, 4IBJ, 4IBK).\n\nFor models and advice please contact Sean Ekins (ekinssean@yahoo.com).", "appendix": "Author contributions\n\n\n\nS.E. and M.C. came up with the general idea for the study based on the published in vitro and in vivo data. All authors contributed to the collaborative writing of this project.\n\n\nCompeting interests\n\n\n\nS.E. works for Collaborations in Chemistry, and consults for Collaborative Drug Discovery Inc.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nDr. Christopher D. Southan, Dr. Peter Madrid and Dr. Nadia Litterman are acknowledged for discussions on Ebola. Biovia are kindly acknowledged for providing Discovery Studio. An earlier preliminary version of this pharmacophore was described previously: http://figshare.com/articles/A_pharmacophore_of_ebola_active_compounds/1190787.\n\n\nSupplementary materials\n\nSupplemental Table 2. FDA drugs and common features pharmacophore. The dataset of 2643 molecules was downloaded from the CDD Public Access (https://www.collaborativedrug.com/pages/public_access) as an sdf and then a 3D database was created in Discovery Studio using FAST conformer generation with up to 255 conformations. The database was searched with the common feature pharmacophore developed from amodiaquine, chloroquine, clomiphene and toremifene. The search 3D database protocol was used with the Fast search method. In some cases the indication for the molecules is not described (ND).\n\nSupplemental Table 3. Microsource Spectrum and common features pharmacophore. The dataset of 2311 molecules was provided by Microsource (http://www.msdiscovery.com/spectrum.html) as an sdf and then a 3D database was created in Discovery Studio using FAST conformer generation with up to 255 conformations. The database was searched with the common feature pharmacophore developed from amodiaquine, chloroquine, clomiphene and toremifene. The search 3D database protocol was used with the Fast search method.\n\nThe 4IBI ligand was removed from the structure and redocked. The closest pose (grey) was ranked 29 with RMSD 3.02A and LibDock score 86.62 when compared to the actual ligand in 4IBI (yellow).\n\n\nReferences\n\nButler D, Morello L: Ebola by the numbers: The size, spread and cost of an outbreak. Nature. 2014; 514(7522): 284–5. PubMed Abstract \| Publisher Full Text\n\nPiot P: Ebola’s perfect storm. Science. 2014; 345(6202): 1221. PubMed Abstract \| Publisher Full Text\n\nTrad MA, Fisher DA, Tambyah PA: Ebola in west Africa. Lancet Infect Dis. 2014; 14(11): 1045. PubMed Abstract \| Publisher Full Text\n\nDhillon RS, Srikrishna D, Sachs J: Controlling Ebola: next steps. Lancet. 2014; 384(9952): 1409–1411. PubMed Abstract \| Publisher Full Text\n\nAnon: Call to action. Nature. 2014; 514(7524): 535–536. PubMed Abstract \| Publisher Full Text\n\nCheck Hayden E: The Ebola questions. Nature. 2014; 514(7524): 554–7. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZheng X, Ekins S, Raufman JP, et al.: Computational models for drug inhibition of the human apical sodium-dependent bile acid transporter. Mol Pharm. 2009; 6(5): 1591–603. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDiao L, Ekins S, Polli JE: Quantitative structure activity relationship for inhibition of human organic cation/carnitine transporter. Mol Pharm. 2010; 7(6): 2120–2131. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEkins S, Diao L, Polli JE: A substrate pharmacophore for the human organic cation/carnitine transporter identifies compounds associated with rhabdomyolysis. Mol Pharm. 2012; 9(4): 905–913. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDong Z, Ekins S, Polli JE: Structure-activity relationship for FDA approved drugs as inhibitors of the human sodium taurocholate cotransporting polypeptide (NTCP). Mol Pharm. 2013; 10(3): 1008–19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDong Z, Ekins S, Polli JE: Quantitative NTCP pharmacophore and lack of association between DILI and NTCP Inhibition. Eur J Pharm Sci. 2014; 66C: 1–9. PubMed Abstract \| Publisher Full Text\n\nKazmi F, Hensley T, Pope C, et al.: Lysosomal sequestration (trapping) of lipophilic amine (cationic amphiphilic) drugs in immortalized human hepatocytes (Fa2N-4 cells). Drug Metab Dispos. 2013; 41(4): 897–905. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNadanaciva S, Lu S, Gebhard DF, et al.: A high content screening assay for identifying lysosomotropic compounds. Toxicol In Vitro. 2011; 25(3): 715–23. PubMed Abstract \| Publisher Full Text\n\nde Wilde AH, Jochmans D, Posthuma CC, et al.: Screening of an FDA-approved compound library identifies four small-molecule inhibitors of Middle East respiratory syndrome coronavirus replication in cell culture. Antimicrob Agents Chemother. 2014; 58(8): 4875–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVincent MJ, Bergeron E, Benjannet S, et al.: Chloroquine is a potent inhibitor of SARS coronavirus infection and spread. Virol J. 2005; 2: 69. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEkins S, Freundlich JS: Computational models for tuberculosis drug discovery. Methods Mol Biol. 2013; 993: 245–62. PubMed Abstract \| Publisher Full Text\n\nEkins S, Freundlich JS, Choi I, et al.: Computational databases, pathway and cheminformatics tools for tuberculosis drug discovery. Trends Microbiol. 2011; 19(2): 65–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEkins S, Williams AJ, Krasowski MD, et al.: In silico repositioning of approved drugs for rare and neglected diseases. Drug Disc Today. 2011; 16(7–8): 298–310. PubMed Abstract \| Publisher Full Text\n\nEkins S, Freundlich JS, Coffee M: Dataset 1 in: A common feature pharmacophore for FDA-approved drugs inhibiting the Ebola virus. F1000Research. 2014. Data Source" }	[ { "id": "6707", "date": "01 Dec 2014", "name": "Gloria Fuentes", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe current Ebola crisis in West Africa has shattered all expectations by continuing to grow months following the initial case. This has stimulated a massive and global emergency response, and it has challenged the health protocols and by extension, the efforts of scientific community.The authors have carried out a computational analysis using several compounds detected in two previous high throughput screens to build a pharmacophore model. The key features of such a model are used to scan databases of small molecules. They have come up with a list of putative inhibitors. Their study will have a stronger scientific impact if the authors could elaborate more in suggestions on how the best ranked compounds will increase the binding affinity, based in the structural model VP30-inhibitors that they have built. In parallel, they observed a highly overlapping between the motifs in the pharmacophore and those found in the crystal structures of several inhibitors of the viral protein 35. Based on this fact, and in their results in an in-silico docking, they propose that the most likely inhibitory mechanism for these compounds is the targeting of the protein-protein interaction involving this protein. In this regard, the authors should extend their study to include different docking protocols, including different programs, in an attempt to verify their results. As they mention in the text (page 4), the redocking of the ligand in 4IBI to the protein does not show the crystal structure binding mode accurately. These different settings could help in a better prediction of the ligand orientations.Concerning the docking and proposed mechanism, I wonder how different the other solved nucleocapsid proteins are from a structural and sequence point of view, in order to make the authors point that VP35 is indeed the target. Would it be possible to explore for surface patches with similar physico-chemical features? In the case of VP30, a potential binding pocket for small-molecule inhibitors has been suggested by Hartlieb et al. (2007). How good or bad the overlapping with the built pharmachophore is for this case?To complete the structural understanding of the action of these compounds, a figure displaying their location on the protein surface as well as the binding site for RNA would clarify their role in the inhibition of protein-protein interactions. In summary, the manuscript describes an interesting and fast approach to identify putative inhibitors for a currently serious target as Ebola virus. Although their results should be experimental validated to confirm their finding, this computational study and further extensions of it are of the great value.", "responses": [ { "c_id": "1116", "date": "12 Dec 2014", "name": "Sean Ekins", "role": "Author Response", "response": "The current Ebola crisis in West Africa has shattered all expectations by continuing to grow months following the initial case. This has stimulated a massive and global emergency response, and it has challenged the health protocols and by extension, the efforts of scientific community.The authors have carried out a computational analysis using several compounds detected in two previous high throughput screens to build a pharmacophore model. The key features of such a model are used to scan databases of small molecules. They have come up with a list of putative inhibitors. Their study will have a stronger scientific impact if the authors could elaborate more in suggestions on how the best ranked compounds will increase the binding affinity, based in the structural model VP30-inhibitors that they have built.Response: To clarify, we have focused on VP35 not VP30. I am not aware of an X-ray structure with ligand bound for VP30. The same type of approach could certainly be pursued with other EBOV targets. We produced a common features pharmacophore for the 4 compounds, and after looking at the VP35 receptor-ligand pharmacophores proposed that there may be some overlap, and then this led to docking the 4 compounds in the X-ray structures. Our intent was not to design molecules but to use the available methods to perhaps infer a potential target/mechanism and then perhaps researchers would want to test the compounds. We do not have access to experimentally test these predictions, but this manuscript may lead to others doing this work perhaps. Whether one wants to use the Libdock score for (absolute) prediction of binding affinity interactions is debatable, rather this approach might help to limit or prioritize which compounds to test. In parallel, they observed a highly overlapping between the motifs in the pharmacophore and those found in the crystal structures of several inhibitors of the viral protein 35. Based on this fact, and in their results in an in-silico docking, they propose that the most likely inhibitory mechanism for these compounds is the targeting of the protein-protein interaction involving this protein. Response: VP35 may be a target for these compounds although we do not discount other targets or non-target related mechanisms. In this regard, the authors should extend their study to include different docking protocols, including different programs, in an attempt to verify their results. As they mention in the text (page 4), the redocking of the ligand in 4IBI to the protein does not show the crystal structure binding mode accurately. These different settings could help in a better prediction of the ligand orientations.Response: As explained earlier, our study is not intended to be an exhaustive evaluation of docking tools, we have used different computational approaches to suggest that the FDA drugs may have a common pharmacophore, which seems to be similar to that of the ligands co-crystallized with VP-35. Finally docking suggests they may fit into the pocket that the co-crystal ligands bind to. The work proposes that the compounds could fit in the binding site, but it is unclear what additional value more docking would add unless we were going to try to predict and then generate the X-ray structure of these FDA drugs. Certainly if experts in docking or crystallography want to pursue this target they can. Concerning the docking and proposed mechanism, I wonder how different the other solved nucleocapsid proteins are from a structural and sequence point of view, in order to make the authors point that VP35 is indeed the target. Would it be possible to explore for surface patches with similar physico-chemical features? In the case of VP30, a potential binding pocket for small-molecule inhibitors has been suggested by Hartlieb et al. (2007). How good or bad the overlapping with the built pharmachophore is for this case?Response: this is indeed a very good point. We are not experts on these proteins. I think the proposed work could be done, the difficulty may be that there is no crystal structure (that I can see) with a ligand bound that would be a useful guide to binding in this pocket and would be essential for a receptor-ligand pharmacophore to be built. To complete the structural understanding of the action of these compounds, a figure displaying their location on the protein surface as well as the binding site for RNA would clarify their role in the inhibition of protein-protein interactions.Response: we have now added Figure S2 which shows the molecules in the context of the full protein. They are in the site suggested in ref 15 as important for the nucleoprotein interaction. In summary, the manuscript describes an interesting and fast approach to identify putative inhibitors for a currently serious target as Ebola virus. Although their results should be experimental validated to confirm their finding, this computational study and further extensions of it are of the great value.Response: Thank you for your suggestions, we agree and would encourage other scientists to test whether these compounds are targeting VP35 or VP 30 as you propose, or having an alternative mechanism. I think we would also be happy to see any of these molecules progress into other animal models of EBOV." } ] }, { "id": "6708", "date": "02 Dec 2014", "name": "Sandeep Chakraborty", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nEkins et al. have presented a crisp and lucid manuscript on a very relevant topic. They have suggested a methodology to extract common features from four approved compounds that have recently been found to work against the Ebola virus (amodiaquine, chloroquine, clomiphene and toremifene), and define a pharma-cophore, which has been used to search databases, and identify further compounds for in vitro and in vivo testing. The in silico methodology described here provides an excellent method to quickly screen known compounds for possible therapies against Ebola in particular, and other viruses in general.Some minor comments.The result that SERMs show lower scores for binding to VP35 is rationalized by the finding that ‘clomiphene and toremifene inhibit EBOV VLP entry with some specificity to GP’1, and therefore does not probably inhibit VP35. ‘Chloroquine to date has not progressed beyond the mouse EBOV model used in these studies.’ This statement is not clear, does it mean that the others have progressed beyond the mouse EBOV model? The first few compounds in Supplemental Table 2 should be part of the main manuscript as a table. Color coding of pharmacophore features should be in Fig 1 too (it comes earlier than Table 1, where it is described). The structures look better with a white background. A 3 Å RMSD for redocking a given ligand is quite high2. The authors should consider the use of other docking methods, as a comparison. A table of Libdock scores would help easily analyze results (with a mention of whether a higher score is better, and the significance of a score).The major concern with the manuscript is the use of proprietary software, and data formats, in the study, which makes it difficult for users to probe the resultant docked structures. Further, non-standard formats are subject to the existence of the company which uses it, and not a given in the future.", "responses": [ { "c_id": "1115", "date": "12 Dec 2014", "name": "Sean Ekins", "role": "Author Response", "response": "Ekins et al. have presented a crisp and lucid manuscript on a very relevant topic. They have suggested a methodology to extract common features from four approved compounds that have recently been found to work against the Ebola virus (amodiaquine, chloroquine, clomiphene and toremifene), and define a pharma-cophore, which has been used to search databases, and identify further compounds for in vitro and in vivo testing. The in silico methodology described here provides an excellent method to quickly screen known compounds for possible therapies against Ebola in particular, and other viruses in general. Response: Thank you for your constructive comments.Some minor comments.The result that SERMs show lower scores for binding to VP35 is rationalized by the finding that ‘clomiphene and toremifene inhibit EBOV VLP entry with some specificity to GP’1, and therefore does not probably inhibit VP35.Response: While the lower docking scores are noted I do not think this necessarily excludes them from inhibiting, as we know docking scores may not be that accurate and in this case, docking was used to answer the question could they fit. It’s pretty clear that a wide variety of drugs could fit based on the binding site size and accessibility. ‘Chloroquine to date has not progressed beyond the mouse EBOV model used in these studies.’ This statement is not clear, does it mean that the others have progressed beyond the mouse EBOV model?Response: From discussions with the author on the paper that described Chloroquine as active versus EBOV in vitro and in mouse, this work has not gone beyond the mouse model of EBOV. The first few compounds in Supplemental Table 2 should be part of the main manuscript as a table.Response: Because this data is available easily on the website, I do not see any benefits of taking these compounds out of this supplemental table and putting them into the body of the manuscript. It might also add more confusion cutting the table up. Color coding of pharmacophore features should be in Fig 1 too (it comes earlier than Table 1, where it is described).Response: Thank you – this has now been added. The structures look better with a white background.Response: I think this is a personal preference, the structures are clear in our opinion with a black background. I have not had this suggestion previously with other publications regarding the background color. A 3 Å RMSD for redocking a given ligand is quite high2. The authors should consider the use of other docking methods, as a comparison.Response: The study was not intended as an exhaustive docking comparison, there are plenty of these in the literature as noted by the reviewer. I agreed the redocking RMSD was high, but I also provided some justification for the result (difficulty of predicting orientations for compounds binding in what is a relatively hydrophobic and shallow pocket). If others want to use different methods and perform a comparison for this target I would be supportive. A table of Libdock scores would help easily analyze results (with a mention of whether a higher score is better, and the significance of a score).Response: The Libdock scores for the best poses are in the ‘4IBI Libdock docking data best poses” file. A higher score is better and this has been added to the results sectionThe major concern with the manuscript is the use of proprietary software, and data formats, in the study, which makes it difficult for users to probe the resultant docked structures. Further, non-standard formats are subject to the existence of the company which uses it, and not a given in the future.Response: All of the models were generated with the proprietary software Discovery studio, and all files have been provided. The comment about such software is true, while I support using open software, I have yet to find an open source pharmacophore tool as good as that in Discovery Studio to date. It is also more convenient to use this software generating pharmacophores, receptor-ligand pharmacophores and docking in the same place. The types of analysis I have described could be repeated with any software, open source or proprietary. My hope is that by making this work openly accessible others will be inspired to pursue computational approaches with EBOV. Perhaps the community could propose the use of standards for open pharmacophore files as well. By publishing in this journal we are making all our data open even though they are in proprietary formats, I do not think this should preclude publication." } ] } ]	1	https://f1000research.com/articles/3-277
https://f1000research.com/articles/3-109/v1	14 May 14	{ "type": "Opinion Article", "title": "The Human Release Hypothesis for biological invasions: human activity as a determinant of the abundance of invasive plant species", "authors": [ "Heike Zimmermann", "Patric Brandt", "Joern Fischer", "Erik Welk", "Henrik von Wehrden", "Patric Brandt", "Joern Fischer", "Erik Welk", "Henrik von Wehrden" ], "abstract": "Research on biological invasions has increased rapidly over the past 30 years, generating numerous explanations of how species become invasive. While the mechanisms of invasive species establishment are well studied, the mechanisms driving abundance patterns (i.e. patterns of population density) remain poorly understood. Invasive species typically have higher abundances in their new environments than in their native ranges, and patterns of invasive species abundance differ between invaded regions. To explain differences in invasive species abundance, we propose the Human Release Hypothesis. In parallel to the established Enemy Release Hypothesis, this hypothesis states that the abundance of invasive species may be partly explained by the level of human activity or landscape maintenance, with intermediate levels of human activity providing optimal conditions for high abundance. The Human Release Hypothesis does not negate other important drivers of species invasions, but rather should be considered as a potentially important additional or complementary mechanism. We illustrate the hypothesis via a case study on an invasive rose species, and hypothesize which locations globally may be most likely to support high abundances of invasive species. We propose that more extensive empirical work on the Human Release Hypothesis could be useful to test its general applicability.", "keywords": [ "Biological invasions can threaten ecosystems1", "economies2", "and human health3. The Scientific Committee on Problems of the Environment (SCOPE) put biological invasions on top of its research agenda in 19834. Since then", "the field of invasion ecology has rapidly gained momentum. The number of publications dealing with biological invasions has increased a hundredfold in less than two decades5. Several journals are partly (e.g. Diversity and Distributions", "Natural Areas Journal) or fully (e.g. Biological Invasions", "Invasive Plant Science and Management", "NeoBiota) devoted to research", "management and policy issues related to invasive species. However", "despite a growing body of knowledge on biological invasions", "difficulties remain in predicting invasion success6." ], "content": "Introduction\n\nBiological invasions can threaten ecosystems1, economies2, and human health3. The Scientific Committee on Problems of the Environment (SCOPE) put biological invasions on top of its research agenda in 19834. Since then, the field of invasion ecology has rapidly gained momentum. The number of publications dealing with biological invasions has increased a hundredfold in less than two decades5. Several journals are partly (e.g. Diversity and Distributions, Natural Areas Journal) or fully (e.g. Biological Invasions, Invasive Plant Science and Management, NeoBiota) devoted to research, management and policy issues related to invasive species. However, despite a growing body of knowledge on biological invasions, difficulties remain in predicting invasion success6.\n\nWithin Europe, the distribution of people is strongly related to the number of alien species. Presumably, this reflects that biological invasions are aided by human transport and that species establishment is facilitated by human disturbance7. Nevertheless, at the global scale, the proportion of widely distributed alien plant species (relative to all species) is far lower in Europe than in North America – despite Europe’s long history of trade and therefore a longer residence time of alien plants8. The observation that Europe serves as a global contributor of alien plant species, whereas North America seems to be a better recipient, has sparked the concept of biological resistance, which explains invasion success or failure in relation to the traits of the native flora9. An additional important consideration, which has not been assessed to date, could be that Europe also has a higher proportion of landscapes that are actively managed by humans than, for example, the Americas, Australia and Africa10. To date, approaches to predict invasion patterns in response to anthropogenic global change have focused on (i) the extent of novel ecosystems11, and (ii) alien species richness12.\n\nIn this paper, we propose that the abundance of an alien species in a given landscape can be (at least partly) explained by the level of active landscape maintenance by humans. We term this hypothesis the Human Release Hypothesis. As discussed in detail below, the Human Release Hypothesis states that the abundance of invasive species may be partly explained by the level of human activity or landscape maintenance, with intermediate levels of human activity providing optimal conditions for high abundance. Unlike the Disturbance Hypothesis and the Intermediate Disturbance Hypothesis, which explain patterns of establishment of invasive species13 and patterns of native species diversity respectively14, the Human Release Hypothesis specifically addresses patterns in the abundance of alien species that are already established in particular areas outside their native ranges.\n\nWe first discuss how the Human Release Hypothesis fits into the context of other key hypotheses in invasion ecology. We then illustrate the hypothesis via a case study on a global invader, the sweetbriar rose (Rosa rubiginosa L.). Finally, we assess how the Human Release Hypothesis may be integrated into biological invasion research, and we hypothesize which locations worldwide may be particularly prone to supporting high abundances of invasive species.\n\n\nThe Human Release Hypothesis\n\nAccording to Richardson et al. (2000)15, an invasive terrestrial plant species is a naturalized alien species that produces reproductive offspring, often in very large numbers, at considerable distance from parent plants, and thus has the potential to spread over extensive areas. A key question in invasion ecology is how the interaction of species traits with environmental characteristics predicts invasion success, including both establishment and abundance in the new environment6.\n\nCatford et al. (2009)16 summarized 29 leading hypotheses predicting invasion success and integrated them into the PAB-framework (Figure 1). This framework considers the size and frequency of introductions (i.e. propagule pressure, P), ecosystem invasibility based on abiotic characteristics of the new environment (A), and biotic characteristics of an invasive species and its recipient community (B). In this framework, human influence on the invasion process is recognized primarily during the establishment stage. For example, human action can increase propagule pressure17 and multiple introduction events make establishment more likely, because species have a higher chance to encounter suitable environmental conditions18. Multiple introductions of the same species also can lead to higher genetic diversity19. However, examples exist of successful invaders with low genetic diversity20, and stemming from single or few introduction events, suggesting that propagule pressure is only one of many variables explaining invasion patterns21.\n\nThe establishment and abundance of invasive plant species are explained by different mechanisms, which have been summarized by Catford et al. (2009)16 in the PAB framework (see text for details). However, the biological characteristics of a given invading species and of its new environment only partly explain the abundance of established invasive populations. We argue that additional insights can be gained via the Human Release Hypotheses, which can complement the existing PAB framework.\n\nWith respect to abiotic conditions, invasion is facilitated if species are pre-adapted to their new environment, for example due to a similar climate in the new environment22. Like propagule pressure, pre-adaption is not a necessary precondition for successful invasion, because climatic niche shifts have been reported for invasive species23. Disturbance events also provide windows of opportunity for invasive species24. Many invasive plant species are adapted to exploit temporarily favourable conditions through their short life cycles, rapid growth, high reproductive allocation, persistent soil seed banks and rapid germination (the Ideal Weed Hypothesis)25.\n\nFinally, biotic characteristics of the recipient community may involve the absence of natural enemies. The Enemy Release Hypothesis explains invasion success as a function of alien species having escaped their natural enemies, allowing them to allocate resources to growth and reproduction rather than defence26. This would make alien plants stronger competitors. In the context of the Intermediate Disturbance Hypothesis, which proposes higher species diversity at intermediate frequencies or intensities of disturbance (see Wilkinson, 1999)14, alien plants are likely to have the greatest impact on community diversity when resources become limited and plant diversity is highest, by co-opting more resources27.\n\nIn parallel to the Enemy Release Hypothesis, here, we propose the Human Release Hypothesis. It describes a situation where alien species have escaped relatively higher levels of human landscape maintenance that is characteristic within their native ranges. Changing patterns of land use are widely recognized to increase opportunities for introduced species to establish and spread28, but already prevailing patterns of land use intensity also should be expected to influence the populations of species – both in their native and introduced ranges. This is because highly intensive land use by humans (such as in many parts of Western Europe) often corresponds to high levels of active landscape maintenance – which translates into little available habitat for both native and introduced species, as well as high levels of active weed control. At the other end of the spectrum of human land use intensity, we hypothesize that pristine natural habitats also offer few windows of opportunity for alien species to establish (the Biotic Resistance Hypothesis)29. Thus, we hypothesize that the abundance of invasive species should be highest in between these two extremes – namely in extensively used landscapes characterized by frequent fallowing, low levels of weed control, high heterogeneity, and many disturbed edges of small farmland patches30. Such landscapes are where “human release” should contribute to optimal conditions for invasive species to establish large populations.\n\nWhile existing hypotheses explain the establishment and naturalization process of invasions, little work has attempted to explain the (potential) abundance of invasive species in their new environments. Part of this gap may be effectively addressed by the Human Release Hypothesis (Figure 1).\n\n\nCase study on an invasive rose\n\nTo illustrate the plausibility of the Human Release Hypothesis, we present findings at two scales on the invasion success of Rosa rubiginosa, a shrub native to Eurasia and invasive in Australia, New Zealand, South Africa, North and South America (see Dataset 1 and Supplementary Figure S1). First, we synthesize previous cross-continental case studies that compared plant performance between invasive populations in Central and Southern Argentina with native populations in Spain and Germany (for more details see Zimmermann et al., 2012)31. Second, we compare climatic conditions as well as land use and human population density between invasive and native R. rubiginosa populations at a global scale. In combination, our findings suggest the Human Release Hypothesis may be a useful complementary hypothesis to other existing hypotheses in invasion biology (Table 1).\n\n(aCavallero & Raffaele 2010, bZimmermann et al. 2010, c2011, d2012, eHirsch et al. 2011, fpresent publication).\n\nRosa rubiginosa has successfully invaded a range of ecosystems within Argentina, covering a major climatic gradient, but exhibiting low levels of genetic diversity32,33 (Figure 2a). Low genetic diversity suggests that multiple introduction events constituting particularly high propagule pressure cannot explain the species' invasion success. Despite lower genetic diversity, populations of R. rubiginosa are considerably smaller in Spain and Germany than in Argentina (Figure 3) – native populations consist of 5 to 20 individuals whereas invasive populations consist of hundreds of individuals31. In addition to propagule pressure, abiotic and biotic variables also cannot fully explain the invasion success of R. rubiginosa. In Argentina, the species neither benefits from favourable soil conditions nor from reduced biotic resistance31.\n\n(a) Genetic diversity in Rosa rubiginosa is higher in its native Spanish and German populations than in the introduced populations in Argentina, suggesting the species did not benefit from multiple introductions (for details see Zimmermann et al. 2010)32. (b) The species does not benefit from a climatic pre-adaptation to the new range. The world map shows the species' climatic niche based on the species’ native distribution (blue) and the invasive distribution (pink). Overlap of climatic niches (purple) is minimal. (c) Rosa rubiginosa appears to benefit from “human release” in its new range. The barplot shows the global proportions of different anthropogenic biomes10 according to the location of invasive and native sweetbriar rose populations. The native range has a larger proportion of residential areas and a higher human population density (log people/km2). Only 0.56% of the invasive range is wildlands, and only 0.03% of the native range.\n\nIn parts of Argentina, single disturbance events have offered windows of opportunity for the species to establish populations, some of which have remained undisturbed for 30 years or longer (a)31,36. The low level of human landscape maintenance means that populations can expand over vast areas and consist of hundreds of individuals (a, here along the whole visible lakeside in Patagonia). (a) For our study area in Patagonia we predicted that 36% of the area (5000 km2) was threatened by R. rubiginosa invasion, across a precipitation gradient from 1400 mm/annum (mountains in the far background) to 600 mm/a36. In Argentina R. rubiginosa shrubs have time to grow to their full size (b), by contrast, many native landscapes are regularly maintained; shrubs are regularly trimmed and mostly grow in hedgerows (c, Germany). Furthermore, in Germany and Spain, fewer habitats are available in landscapes dominated by agriculture and urban areas (d, Spain).\n\nMoreover, a global climatic analysis shows that R. rubiginosa also does not depend or benefit from pre-adaptation to the climate of its new environment (Figure 2b). We developed two climatic envelope models based on BioClim parameters and the occurrence of native and invasive populations respectively using the maximum entropy method34 (MAXENT, see Appendix 1 and 2 in the Supplementary material). We detected a significant differentiation of realized niches between invasive and native populations (Schoener’s D=0.31, p<0.0001; Figure 2b). Furthermore, back-projection of the climatic niche based on invasive populations points to a southern European origin. However, genetic analyses tracked the native origin of invasive Argentinean, Chilean, Australian and New Zealand populations to Central Europe32,33. Key climatic predictors therefore do not point to a climatic advantage in the invasive range, but instead indicate that R. rubiginosa is able to thrive under a wide range of conditions (Supplementary Figure S2 and Supplementary Figure S3).\n\nThe Ideal Weed and Disturbance Hypotheses (Table 1) partly explain the invasion success of R. rubiginosa in Argentina31,35,36. However, the Enemy Release Hypothesis failed to explain abundance patterns – natural enemies appeared equally harmful to the species in the native and introduced ranges31 (Table 1). By contrast, in the invasive range, anthropogenic disturbances such as logging and burning create windows of opportunities for the rose to establish, but just as importantly, disturbance events are then followed by decades of abandonment that enable the species to become abundant.\n\nHaving considered a wide range of existing hypotheses (Table 1), we found that additional insights into the invasion patterns of R. rubiginosa may be gained by the Human Release Hypothesis. This is because a key difference between native and introduced environments appears to be the level of active landscape maintenance. In the case study, we observed frequent trimming or removal of individuals only in Spain and Germany and not in Argentina, and individuals and populations in Argentina were significantly older than their native counterparts31,36. At the global scale, our analysis revealed a similar pattern (albeit at a coarser resolution; 2.5 × 2.5 arc min, Figure 2c). Native R. rubiginosa populations occur in areas with higher proportions of cropland, residential areas and human population densities than invasive populations (Figure 2c). These conditions very likely correspond to a high degree of landscape maintenance, and hence little available habitat for R. rubiginosa in its native range.\n\n\n\n\nIntegrating the Human Release Hypothesis with other explanations\n\nA key premise of this paper is that existing hypotheses that predict invasion success can be effectively complemented by the Human Release Hypothesis (Figure 1). Our own data, of course, focused only on one species – which is enough to pose a hypothesis, but far too little to test its general usefulness. To that end, we see two research priorities that should be addressed to further scrutinize the Human Release Hypothesis so that, if appropriate, it can be integrated into invasive species management. First, additional species should be studied in both their native ranges and in different parts of their introduced ranges. Such comparisons would be useful to test the drivers of invasive species abundance and to validate (or refute) invasion patterns derived from modelling approaches11,12. An important first clue that the Human Release Hypothesis may be relevant could be whether invasive individuals of a given perennial species are significantly older than individuals within the native range. Second, it may be useful to further investigate the relationship between landscape maintenance and human land use intensity, how it manifests in different regions, and if generalizations are possible at the global scale. The frequency of weeding and trimming, as well as the prevalence of fallowing, are just two of many potential indicators for the level of active landscape maintenance.\n\nEvidently, the Human Release Hypothesis is still in its infancy, and it would be unwise to make bold management recommendations on its basis. Based on our analysis to date, preliminary insights that are relevant to managing invasive species are: (i) sparsely populated areas may face a higher risk of biological invasions than more densely populated areas; (ii) extensively managed rangelands may be more susceptible to high abundances of invasive species than intensively managed croplands; and (iii) high abundances of invasive species at landscape and regional scales could be facilitated by long periods of fallowing or land abandonment37.\n\n\nData availability\n\nfigshare: Dataset 1. Rosa rubiginosa L. occurrence data (occurrences_R.rubiginosa.csv, 416 kb). Doi: 10.6084/m9.figshare.100206738", "appendix": "Author contributions\n\n\n\nHZ and HvW conceived the study. HvW and PB performed the climatic niche model and PB performed the climatic niche equivalency test. JF and HvW contributed substantially to the framing of the manuscript. EW compiled the geographic distribution of the study species. HZ wrote the first draft of the manuscript and contributed to the data analysis and data collection, and all authors contributed substantially to revisions.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was funded by a Leuphana small research grant 73000787 (HZ) and through a Sofja Kovalevskaja Award by the Alexander von Humboldt Foundation (JF).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe Human Release Hypothesis evolved during previous studies by H. Zimmermann, and we thank all co-authors involved in previous publications: D. Bran, M.A. Damascos, I. Hensen, H. Hirsch, D. Renison, C.M. Ritz, V. Wissemann, and K. Wesche.\n\n\nSupplementary material\n\nhttps://f1000researchdata.s3.amazonaws.com/supplementary/3740/7d02646a-def8-4980-adf8-fd60fb4d75a5.pdf\n\n\nReferences\n\nCrooks JA: Characterizing ecosystem-level consequences of biological invasions: the role of ecosystem engineers. Oikos. 2002; 97(2): 153–66. Publisher Full Text\n\nBorn W, Rauschmayer F, Bräuer I: Economic evaluation of biological invasions—a survey. Ecol Econ. 2005; 55(3): 321–36. Publisher Full Text\n\nMack RN, Smith MC: Invasive plants as catalysts for the spread of human parasites. NeoBiota. 2011; 9: 13–29. Publisher Full Text\n\nDrake J, Mooney HA, di Castri F, et al.: Biological invasions: a global perspective. SCOPE 37. Chichester, UK: John Wiley and Sons; 1989. Reference Source\n\nRicciardi A, MacIsaac H: In Retrospect: The book that began invasion ecology. Nature. 2008; 452: 34. Publisher Full Text\n\nKueffer C, Pyšek P, Richardson DM: Integrative invasion science: model systems, multi-site studies, focused meta-analysis and invasion syndromes. New Phytol. 2013; 200(3): 615–33. PubMed Abstract \| Publisher Full Text\n\nPyšek P, Jarošík V, Hulme PE, et al.: Disentangling the role of environmental and human pressures on biological invasions across Europe. Proc Natl Acad Sci U S A. 2010; 107(27): 12157–62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStohlgren TJ, Pyšek P, Kartesz J, et al.: Widespread plant species: natives versus aliens in our changing world. Biol Invasions. 2011; 13(9): 1931–44. Publisher Full Text\n\nDi Castri F: History of biological invasions with special emphasis on the Old World. In: Drake JA, Mooney HA, di Castri F, Groves RH, Kruger F, Rejmánek M, et al., editors. Biological invasions: a global perspective. Chichester, UK: John Wiley and Sons; 1989. p. 1–30. 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Publisher Full Text\n\nRichardson DM, Pyšek P, Rejmánek M, et al.: Naturalization and invasion of alien plants: concepts and definitions. Divers Distrib. 2000; 6(2): 93–107. Publisher Full Text\n\nCatford JA, Jansson R, Nilsson C: Reducing redundancy in invasion ecology by integrating hypotheses into a single theoretical framework. Divers Distrib. 2009; 15(1): 22–40. Publisher Full Text\n\nLevine JM, Adler PB, Yelenik SG: A meta-analysis of biotic resistance to exotic plant invasions. Ecol Lett. 2004; 7(10): 975–89. Publisher Full Text\n\nLockwood JL, Cassey P, Blackburn T: The role of propagule pressure in explaining species invasions. Trends Ecol Evol. 2005; 20(5): 223–8. PubMed Abstract \| Publisher Full Text\n\nNovak S, Mack R: Genetic bottlenecks in alien plant species: influence of mating systems and introduction dynamics. In: Sax D, Stachowicz J, Gaines S, editors. Species Invasions: Insights into Ecology, Evolution, and Biogeography. Sunderland, Massachusetts: Sinauer Associates; 2005. p. 201–8. Reference Source\n\nLoomis ES, Fishman L: A continent-wide clone: population genetic variation of the invasive plant Hieracium aurantiacum (Orange Hawkweed; Asteraceae) in North America. Int J Plant Sci. 2009; 170(6): 759–65. Publisher Full Text\n\nNuñez MA, Moretti A, Simberloff D: Propagule pressure hypothesis not supported by an 80–year experiment on woody species invasion. Oikos. 2011; 120(9): 1311–1316. Publisher Full Text\n\nWelk E, Schubert K, Hoffmann M: Present and potential distribution of invasive garlic mustard (Alliaria petiolata) in North America. Divers Distrib. 2002; 8(4): 219–33. Publisher Full Text\n\nBroennimann O, Treier UA, Müller-Schärer H, et al.: Evidence of climatic niche shift during biological invasion. Ecol Lett. 2007; 10(8): 701–9. PubMed Abstract \| Publisher Full Text\n\nHobbs RJ, Huenneke LF: Disturbance, diversity, and invasion: implications for Conservation. Conserv Biol. 1992; 6(3): 324–37. Publisher Full Text\n\nRejmánek M, Richardson DM: What attributes make some plant species more invasive? Ecology. 1996; 77(6): 1655–61. Publisher Full Text\n\nKeane RM, Crawley MJ: Exotic plant invasions and the enemy release hypothesis. Trends Ecol Evol. 2002; 17(4): 164–70. Publisher Full Text\n\nCatford JA, Daehler CC, Murphy HT, et al.: The intermediate disturbance hypothesis and plant invasions: Implications for species richness and management. Perspect Plant Ecol Evol Syst. 2012; 14(3): 231–41. Publisher Full Text\n\nHobbs R: Land-use changes and invasions. In: Mooney HA, Hobbs RJ, editors. Invasive species in a changing world. Washington DC: Island Press; 2000. p. 55–64. Reference Source\n\nKennedy TA, Naeem S, Howe KM, et al.: Biodiversity as a barrier to ecological invasion. Nature. 2002; 417(6889): 636–8. PubMed Abstract \| Publisher Full Text\n\nBartomeus I, Sol D, Pino J, et al.: Deconstructing the native–exotic richness relationship in plants. Glob Ecol Biogeogr. 2012; 21(5): 524–33. Publisher Full Text\n\nZimmermann H, von Wehrden H, Renison D, et al.: Shrub management is the principal driver of differing population sizes between native and invasive populations of Rosa rubiginosa L. Biol Invasions. 2012; 14(10): 2141–57. Publisher Full Text\n\nZimmermann H, Ritz CM, Hirsch H, et al.: Highly reduced genetic diversity of Rosa rubiginosa L. populations in the invasive range. Int J Plant Sci. 2010; 171(4): 435–46. Publisher Full Text\n\nHirsch H, Zimmermann H, Ritz CM, et al.: Tracking the origin of invasive Rosa rubiginosa populations in Argentina. Int J Plant Sci. 2011; 172(4): 530–40. Publisher Full Text\n\nElith J, Phillips SJ, Hastie T, et al.: A statistical explanation of MaxEnt for ecologists. Divers Distrib. 2011; 17(1): 43–57. Publisher Full Text\n\nCavallero L, Raffaele E: Fire enhances the “competition-free” space of an invader shrub: Rosa rubiginosa in northwestern Patagonia. Biol Invasions. 2010; 12(10): 3395–404. Publisher Full Text\n\nZimmermann H, von Wehrden H, Damascos MA, et al.: Habitat invasion risk assessment based on Landsat 5 data, exemplified by the shrub Rosa rubiginosa in southern Argentina. Austral Ecol. 2011; 36(7): 870–80. Publisher Full Text\n\nCramer VA, Hobbs RJ, Standish RJ: What’s new about old fields? Land abandonment and ecosystem assembly. Trends Ecol Evol. 2008; 23(2): 104–12. PubMed Abstract \| Publisher Full Text\n\nZimmerman H, Brandt P, Fischer J, et al.: Dataset 1. Rosa rubiginosa L. occurrence data (occurrences_R.rubiginosa.csv, 416 kb). figshare. 2014. Data Source" }	[ { "id": "4769", "date": "23 May 2014", "name": "Christoph Kueffer", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting concept paper. It touches upon two timely research problems in invasion science: (i) how to better incorporate the role of humans into invasion theory, and (ii) how to improve the predictability of the abundance of invasive alien species (instead of only occurrence). The key idea of the proposed ‘human release hypothesis’ seems that land management intensity – especially of abandoned or extensively used habitat – can explain differences in the abundance of a species between its native and alien ranges because the abundance of the species might be reduced in the native range through more intensive management of such land and associated cutting of the species. This is an interesting idea because it states that the presence of humans and their effects on landscapes can reduce invasion spread in opposition to traditional thinking that sees human land use mainly as a driver of invasions. The authors propose for instance that differences in land management coverage and intensity might explain why Europe is less invaded than regions with a higher proportion of wildlands such as North America or Australia.The argumentation of the article fits also well with recent thinking in biodiversity conservation that emphasizes that permanent conservation intervention will in the future increasingly be necessary to maintain threatened biodiversity and ecosystem services in an era of global change; and that this will require building on synergies with other land use practices (such as weeding through agricultural practices in buffer zones around protected areas).", "responses": [] }, { "id": "6008", "date": "02 Sep 2014", "name": "Melisa Giorgis", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript presents a simple and interesting hypothesis about how human activities could drive the increase in abundance of invasive species. It is based on two important observations; 1. Invasive species have in general higher abundances in their new environment than in the native ranges, and 2. The patterns of invasive species differ between invaded regions. The authors realized that human land management activities explain the difference in abundance of species, between both the invader and native range and between different invader regions. Specifically they put a new role for “human activities” into the second stage of plant invasion (Diez & Edwards, 2006). I think that it is an interesting hypothesis, which provides a new vision and background for future research on invasion ecology and conservation management. Finally, I have some suggestions that may help to a better understanding of this article and future development. The need for a clear definition of “active landscape maintenance by human”. The authors at the end mention the frequency of weeding and trimming, as potential indicators of active landscape maintenance. But could human maintenance be defined as any human activity developed in order to sustain the same physiognomy, structure, floristic composition or/and biomass? “Active landscape maintenance” is for me too general. If active landscapes involve just the maintenance of biomass, it could be defined as “disturbance” in the context of “intermediate hypothesis”. But perhaps it might be more than just biomass. Did this hypothesis explain the species abundance in both the native and the invaded range? Please check the third paragraph in the introduction with the third paragraph in the page four. From the manuscript I understand that it explains the abundance in both situations. In the second paragraph of the introduction: I don’t find the aim of the last line. Moreover, the author could improve the first paragraph after the subtitle The Human Release Hypothesis, because I also don’t understand the logic among that paragraph. It seems to be two important sentences (important), but I don’t find any cohesion between them. It is also quite hard to understand what the paragraph aims for. Future context. 1; how this hypothesis works for different life forms (competitor; stress-tolerator; ruderal plant strategy). I find really interesting thinking of this. Maybe the context of Fig. 1 of Diez and Edwards (2006) is a good scene. 2; the manuscript focus on Europe as the principal and the only example, but maybe Asia is another possible example. On one hand it provides a great amount of invasive species and on the other hand it has a higher proportion of dense settlements. Species like Cotoneaster, Ligustrum and Pyracantha that are native from Asia are the most invasive species in the earth and cover at least in Argentine, an significant portion of landscape.", "responses": [ { "c_id": "1114", "date": "12 Dec 2014", "name": "Heike Zimmermann", "role": "Author Response", "response": "Thank you for your thorough and helpful review. We have incorporated all the minor comments concerning typos and wording. For our response to the mayor comments please see below: We included a more detailed explanation of landscape maintenance. The key difference between \"disturbance\" and \"maintenance\" is the time scale. Disturbance can be a single event but maintenance is defined as \"work that is done to KEEP something in good conditions\".See Introduction: \"We define intermediate levels of human activity as activity patterns defined by sporadic disturbance events that are followed by long periods lacking active management, such as fallowing or abandonment. In contrast, regions with high levels of human activity frequently experience active management, such as weeding, hedge trimming or mowing of field margins.\" See section The Human Release Hypothesis in the context of other invasion hypotheses: \"Thus, we hypothesize that the abundance of invasive species should be highest in between these two extremes – namely in extensively used landscapes characterized by frequent fallowing, low levels of weed control, high heterogeneity, and many disturbed edges of small farmland patches32. Such landscapes are where “human release” should contribute to optimal conditions for invasive species to establish large populations.\" Yes, it does explain the abundance in both ranges. We clarified this now further in the Introduction: \"Finally, we propose that the Human Release Hypothesis can also explain why some species that are highly abundant in their invasive range have relatively low abundance in their native range.\" We re-wrote this paragraph and hope it is now comprehensible:\"To date, extensive data on the abundance of invasive alien species is widely lacking. Existing approaches to predict invasion patterns in response to anthropogenic global change have focused primarily on the development of novel ecosystems11 and alien species richness12. Based on this, it is now widely acknowledged that systems containing high numbers of alien species tend to be those created and sustained by humans.\" Future context. We now included a paragraph on how our hypothesis applies to different life strategies, and we encourage to investigate how comparisons between species from Asia and their invasive range could fit to our hypothesis. See section The Human Release Hypothesis in the context of other invasion hypotheses: \"Disturbance events also provide windows of opportunity for invasive species26 and are often the result of human activity. Many invasive plant species are adapted to exploit temporarily favourable conditions through their short life cycles, rapid growth, high reproductive allocation, persistent soil seed banks and rapid germination (the Ideal Weed Hypothesis)27. All these traits are also of advantage in systems where frequent weeding or mowing is practiced. Therefore, species pursuing this competitive ruderal strategy could profit twofold from Human Release.\"See last section: \"We generated our hypothesis based on findings in Europe, however many invasive plant species on the American continent originate from Asia42,43, thus it would be interesting to test our hypothesis based on land use patterns from these regions. \"" } ] } ]	1	https://f1000research.com/articles/3-109
https://f1000research.com/articles/3-304/v1	12 Dec 14	{ "type": "Review", "title": "Does apical membrane GLUT2 have a role in intestinal glucose uptake?", "authors": [ "Richard J. Naftalin" ], "abstract": "It has been proposed that the non-saturable component of intestinal glucose absorption, apparent following prolonged exposure to high intraluminal glucose concentrations, is mediated via the low affinity glucose and fructose transporter, GLUT2, upregulated within the small intestinal apical border.The evidence that the non-saturable transport component is mediated via an apical membrane sugar transporter is that it is inhibited by phloretin, after exposure to phloridzin. Since the other apical membrane sugar transporter, GLUT5, is insensitive to inhibition by either cytochalasin B, or phloretin, GLUT2 was deduced to be the low affinity sugar transport route.As in its uninhibited state, polarized intestinal glucose absorption depends both on coupled entry of glucose and sodium across the brush border membrane and on the enterocyte cytosolic glucose concentration exceeding that in both luminal and submucosal interstitial fluids, upregulation of GLUT2 within the intestinal brush border will usually stimulate downhill glucose reflux to the intestinal lumen from the enterocytes; thereby reducing, rather than enhancing net glucose absorption across the luminal surface.These states are simulated with a computer model generating solutions to the differential equations for glucose, Na and water flows between luminal, cell, interstitial and capillary compartments. The model demonstrates that uphill glucose transport via SGLT1 into enterocytes, when short-circuited by any passive glucose carrier in the apical membrane, such as GLUT2, will reduce transcellular glucose absorption and thereby lead to increased paracellular flow. The model also illustrates that apical GLUT2 may usefully act as an osmoregulator to prevent excessive enterocyte volume change with altered luminal glucose concentrations.", "keywords": [ "Intestinal glucose absorption has been studied for more than a century and still remains controversial. During the last fifty years the main research thrust has been to identify and characterize the individual transport components within the intestinal epithelium. This progressively reductivist approach has been very successful: we have a comprehensive knowledge of the nature of the driving forces generating sugar absorption", "the specificity range of the sugar transporters involved", "their sites of activity within the enterocytes and of how the individual transport processes function at a molecular level1–3. Less clear is how the intestine functions as a working ensemble to absorb glucose over the wide range of luminal concentrations occurring within the small intestine and how this process is controlled", "both in the short and long-term. These uncertainties arise from the multiplicity and complexity of interactive processes and lack of a comprehensive model permitting an integrated view of intestinal glucose uptake." ], "content": "Introduction\n\nIntestinal glucose absorption has been studied for more than a century and still remains controversial. During the last fifty years the main research thrust has been to identify and characterize the individual transport components within the intestinal epithelium. This progressively reductivist approach has been very successful: we have a comprehensive knowledge of the nature of the driving forces generating sugar absorption; the specificity range of the sugar transporters involved; their sites of activity within the enterocytes and of how the individual transport processes function at a molecular level1–3. Less clear is how the intestine functions as a working ensemble to absorb glucose over the wide range of luminal concentrations occurring within the small intestine and how this process is controlled, both in the short and long-term. These uncertainties arise from the multiplicity and complexity of interactive processes and lack of a comprehensive model permitting an integrated view of intestinal glucose uptake.\n\nThe early opinion on intestinal glucose transport was that stereospecific electrogenic active transcellular transport process coexisted with a variable non-specific paracellular diffusive flux4–8. Intestinal glucose absorption entails specific sodium-dependent hexose interactions with jejunal and ileal enterocyte glucose transporters in the apical and sodium-independent passive downhill transport via basal-lateral membranes and transit by solvent drag via non-selective paracellular pathways, generated by electro-osmotic flow of Na+ and water7,9,10, or by paracellular passive diffusion down the glucose concentration gradient existing between the intestinal lumen and lamina propria11,12. This diffusive route permits non-specific transport of L-glucose, D-rhamnose, or mannitol, as well as D-glucose at rates that are correlated with net fluid transport13. The general consensus was that at around a luminal glucose ≈ 25 mM the active and passive components are about equal and above this passive absorption becomes dominant (Figure 1).\n\nThe tissue in panel A has low apical GLUT2 and GLUT5 activity and low capillary permeability and perfusion rates (clearance). In panel B the tissue apical membrane GLUT2 activity is increased by 4-fold above that in panel A, capillary perfusion is unchanged. In panel C, the apical GLUT2 activity is the same as in panel A, but capillary clearance is increased by 10-fold. In panel D, the apical GLUT2 is raised, as in panel B and the capillary clearance raised, as in panel C.\n\nThe rates of glucose uptake are normalized relative to the rate of SGLT1 glucose uptake (panel A). Altering either GLUT2, or capillary clearance have negligible effects on glucose inflow via SGLT1. However, after raising the apical GLUT2 activity, the steady state glucose concentration within the cytosol decreases from 68 to 52 mM (c.f. Panels A and C). On raising capillary clearance, the steady state of cytosolic glucose concentration also decreases (c.f. Panel A versus Panel C and Panel B versus Panel D).\n\nRaising capillary glucose clearance increases the rate of glucose inflow from the interstitial to capillary fluid by fourteen fold (c.f. Panel A and C). These changes are accompanied by decreased interstitial fluid glucose from 52 to 40 mM and reductions in the mean capillary glucose from 23 to 18 mM. Reduced interstitial glucose concentrations reverse the direction of the glucose gradient across the paracellular pathway from -2 to + 10 mM. Thus raising the capillary clearance of glucose, reverses the direction of paracellular glucose flow from (-0.38) to (+2.46) and increases the net glucose inflow across the luminal surface from (0.22 to 3.23).\n\nAlthough raising apical membrane GLUT2 activity by fourfold reduces net glucose influx across the apical border from 0.63 to 0.15, it also indirectly leads to an increase in paracellular glucose flux and thereby causes a slight increase in net glucose flux across the luminal border.\n\nWhen capillary clearance is raised, either by enhanced perfusion rates, or increased endothelial permeability, increasing apical membrane GLUT2 enhances apical membrane glucose reflux from -0.14 to -0.31. This has no significant effect on glucose flow from the interstitial to capillary fluid. (c.f. panel C and D).\n\nThis dual transport model explained why the apparent affinity of total net glucose uptake is much less, Km > 62.3±3.2 mM than the Km obtained for electrogenic glucose transport (Km = 17.9±0.4 mM); and why phloridzin, a blocker of Na-coupled glucose transport via SGLT1 at the luminal surface, affects mainly electrogenic transport, but not transport via the paracellular route4.\n\nParsons and colleagues14,15 were amongst the first to postulate parallel active and passive absorptive processes in the luminal surface intestinal membrane.\n\nKellett and colleagues1,16,17 later proposed that when luminal glucose is raised above 15 mM, that the non-saturable absorptive component, instead of being via the paracellular route is due to influx via a low affinity glucose transporter, GLUT2, whose presence is regulated within jejunal and ileal enterocytes apical membranes. The salient experimental evidence supporting this view is that the “non-saturable” component of glucose absorption is inhibited by either high phloretin (0.75–1 mM), or high cytochalasin B (0.2 mM) concentrations, both of which inhibit GLUT2 and neither of which inhibit GLUT5.\n\nUsing a sigmoid curve fit, Kellett and Helliwell1 obtained a Km of the phloretin-sensitive component “similar” to that of GLUT2, 56±14 mM; n=1.6±0.4. They argued that GLUT2 is the most likely route for this low affinity transport, since it also transports fructose. Later reports showed that artificial sweeteners e.g. aspartame, sucralose and saccharin in parallel with an increase in intracellular calcium, increase the rate of glucose absorption, by increasing brush border GLUT218 and this in turn increased release of several incretins gluco-insulinotropic peptide(GIP); glucagon- like peptide (GLP-1) and peptide tyrosine-tyrosine (PYY) from enteroendocrine cells19.\n\nAlthough these arguments seem plausible, there are several reasons to question the assertion that apical membrane GLUT2 mediates the low affinity component of intestinal D-glucose absorption. Many studies have shown that the low affinity glucose absorptive route has low specificity- it can transport sugars e.g. L-glucose or rhamnose, or low molecular weight solutes, such as Cr-EDTA, or mannitol, that are not transported by any GLUTs13. Thus the explanation that GLUT2 is the sole mediator of the low affinity sugar transport route does not explain transport of these paracellular markers without any affinity for sugar transporters.\n\nThe Km of GLUT2 has been measured as approximately 17 mM20,21, this value is much lower than the very high Km 56±14 mM observed by Kellett & Helliwell (2000)1. Additionally, at luminal glucose concentrations > 50 mM absorption linearly correlates with luminal concentration; i.e. is not saturable8. Thus the high Km of the “phloretin-sensitive” component does not necessarily signify glucose transport via a low affinity glucose transporter.\n\nFurthermore, phloretin- is not uniquely specific as a glucose-transporter inhibitor. Phloretin also blocks chloride, or aquaporin water channels, or urea transporter mediated urea and water transport, probably by intercalating with the lipid membrane and consequently may also inhibit solute and water paracellular transport22,23. Hence, a transport process blocked by high concentrations of phloretin or cytochalasin B need not imply that the inhibited flow is mediated via apical membrane GLUT2.\n\nIn contrast to Kellett and colleagues’ claims, other studies with GLUT2 knock out (KO) mice have shown that GLUT2 makes no substantial contribution to net glucose absorption and furthermore that D-glucose accumulation in enterocytes is increased in GLUT2 KO mice20,24. This increase can in part be ascribed to loss of GLUT2 mediated transport activity from the baso-lateral membranes. Doubts have also been raised as to whether GLUT2 is expressed at all in the intestinal apical membranes25. Roder et al.24, were unable to detect significant levels of GLUT2 within the intestinal brush borders of wild type mice. Additionally, in humans there is an absence of any detectible increased response to artificial sweeteners with relation to any increased sugar uptake, or incretin release26,27.\n\nHowever, Kellett28, has responded to some of these arguments, suggesting that the mice used in these KO studies were not optimally prepared. Starvation leads to loss of both intestinal GLUT2 apical protein and GLUT2 mRNA, whereas re-feeding after a period of starvation leads to a rapid increase in both apical GLUT2 expression and to GLUT2 mRNA expression within the intestine29.\n\nThe later results reported by Brot-Laroche’s group appear to conflict with some earlier data from her laboratory showing that semi-starvation increased the Vmax and Km of D-glucose uptake into guinea pig jejunal brush border membrane vesicles (BBMV)30. Starvation was postulated to induce a secondary low affinity glucose transport system. Additional studies revealed that phloretin (0.25 mM) enhanced the initial rate of D-glucose uptake by 15% into guinea-pig BBMV. Application of Student’s two-tailed t–test shows that this increase is significant (P < 0.012). Cytochalasin B (0.1 mM) inhibited D-glucose (10 mM) uptake by 38% (p < 0.0001), but had negligible effects on SGLT1 specific α-methyl-D-glucoside uptake31. The earlier results imply that phloretin enhances, rather than inhibits, the low affinity D-glucose transport in BBMV, as was later asserted1,18.\n\nRecent live imaging studies indicate that GLUT2 is a variable presence within the apical membrane32; its trafficking being dependent on signals induced by high intracellular glucose concentrations.\n\n\nAnalysis\n\nAlthough upregulation of apical membrane GLUT2 is a feature of raised luminal D-glucose concentrations, it is far from clear, as contended, that this leads to enhanced net glucose transport1,17. Since both active (SGLT1) and passive glucose transporter (GLUTs 2 and 5) elements are present within the brush border membranes, the kinetics of net glucose flow across the brush border ensemble will depend both on the variable glucose concentrations in the adjacent luminal and cytosolic compartments and the relative proportions of active and passive transport components and the area of absorbing intestinal surface exposed to glucose. The steady state cytosolic and interstitial glucose concentrations are also reliant upon the concentration dependence of flows across the baso-lateral membrane into the interstitial fluid and between the luminal fluid and interstitial fluid via the intercellular junctions. GLUT2 (Km ≈ 17 mM) is the main transporter for glucose movement across the basal-lateral membranes33,34.\n\nThe apparent transport parameters (Km and Vmax)4–6 obtained. In intestinal enterocytes in situ, where flows with varying luminal glucose concentrations are normally measured in steady state, have scant resemblance to those obtained in zero-trans conditions with isolated membrane vesicles or oocytes.\n\nUphill glucose transport via the apical membrane sodium-glucose cotransporter SGLT1 generates polarized sugar flow, causing the intracellular glucose concentration to increase: eventually the cytosolic and also interstitial glucose concentrations may exceed the luminal concentration35. Once these conditions are met, glucose will reflux back into the intestinal lumen via passive transporters in the apical membrane, or via the tight junction (Figure 1A). If the Vmax of the passive apical membrane glucose transporters is raised, then owing to enhanced glucose reflux via GLUT2, net glucose influx across the apical membrane will be reduced. However, net glucose uptake across the luminal surface, including the paracellular pathways may be augmented. This increase in paracellular glucose flow arises from decreased transcellular flow. The resulting slight decrease in interstitial fluid glucose concentration increases the gradient between the intestinal lumen and interstitial fluid, (Figure 1B) and Figure 3(A–C).\n\nGlucose influx across the apical membrane remains polarized over a very wide concentration range, due to the very low affinity for glucose at the export site of SGLT135,36. Thus when both the cytosolic and the interstitial glucose concentrations are close to GLUT2 saturation levels; i.e. D-glucose > 30 mM, the resistance to glucose outflow across the baso-lateral membrane will increase. Consequently, cytosolic concentration may increase disproportionally as luminal glucose concentrations rise, since the apical and baso-lateral membranes may act as a double membrane rectifier to glucose flow37. This promotes non-linear glucose accumulations in the intermediate cytosol between the apical and baso-lateral membranes11,38,39 (Figure 1A).\n\nGlucose flow from the interstitial fluid into the villus capillaries depends on the glucose diffusion between the interstitial fluid and the mean capillary luminal concentration. The mean capillary luminal glucose concentration is a complex non-linear function of the glucose permeability of the capillary membranes, the systemic arterial glucose concentration and the capillary flow rate11. The boundary conditions of this flow network determine the steady-state glucose concentrations within all the intermediate compartments.\n\nRaising intestinal luminal glucose above 30 mM results in increased superior mesenteric arterial flow from around 1000–2500 ml min-140–42. Raised capillary glucose clearance will reduce the interstitial glucose concentration, thereby also reducing cytosolic glucose concentration, thus increasing net influx across the luminal surface, whilst reducing glucose reflux both via brush border passive transporters and via the paracellular pathway. The model simulates all these conditions as seen by comparing Figures 1A and 1B with Figures 1C and 1D.\n\nHowever, even with high rates of vascular perfusion, the interstitial glucose concentration approximates to that of the luminal concentration. Consequently, as luminal glucose concentrations are raised, even although interstitial capillary glucose clearance is increased, the enterocyte cytosolic concentrations continuously rises, (Figure 2C and 2D)11.\n\nThe simulations show the glucose fluxes via apical SGLT1 (blue); apical GLUT2 (red); paracellular pathway (green); the total transluminal membrane, (SGLT1 + GLUT2 + paracellular fluxes), (black) and interstitial to capillary flow (pink crosses) inset on the black square. The main effect of increasing GLUT2 is to cause a negative glucose flux (backflux) via GLUT2 (Panel B). This is accompanied by a increased paracellular flux without any significant change in net transluminal or transepithelial glucose flux. The point at which paracellular glucose flux and SGLT1 flux are equal lies between 20 and 30 mM as has been previously observed4–6. This value is used as one of the key registration points for the model.\n\nThe cytosolic (red) interstitial (blue) and mean capillary glucose concentrations (black) and enterocyte volume per unit weight of tissue (green) are shown in panel C with zero apical GLUT2 and in Panel D with GLUT2 Vmax = 2.\n\nIncreased apical GLUT2 activity decreases cytosolic glucose concentration (panel D). With rising luminal glucose concentration raised GLUT2 activity prevents the non-linear increase in enterocyte volume seen with zero GLUT2 (Panel C).\n\nThe effects of varying paracellular glucose permeability Pglc from 0 to 0.02 cm s-1 are shown in Figure 3A increasing Pglc on paracellular glucose flux (Blue) .As Pglc is increased from zero the point of equality of paracellular glucose flux Jglpc with glucose flux via SGLT1 decreases from infinity at Pglc = 0 to around 20–30 mM luminal glucose when Pglc = 0.01–0.02 cm s-1.\n\nIncreases Pglc raises interstitial glucose concentrations 3C (blue) and in parallel, cysosolic concentrations Figure 3C (red). The reduction in glucose gradient across the basolateral membrane with raised Pglc reduces and then reverses glucose flux via GLUT2 (Figure 3B).\n\nCapillary clearance of glucose is a key factor affecting net intestinal glucose absorption at the intestinal border, as submucosal capillary glucose concentration rapidly equilibrates with that in the interstitial solution15. Intestinal glucose clearance depends on the local blood flow rate, determined by the superior mesenteric arterial (SMA) pressure and its compliance and also the mean glucose concentration difference between the villus capillaries and the interstitial solution (Figure 1A–D). Thus, as can be seen by comparing Figures 1A and 1B with Figures 1C and 1D, increasing the capillary clearance reduces the interstitial glucose concentration, thereby increasing the glucose gradients across the baso-lateral membranes and between the luminal and interstitial solutions, thereby enhancing absorptive flux.\n\nWith constant high capillary glucose clearance, increasing apical GLUT2 activity, whilst enhancing glucose backflux across the apical membrane, also increases paracellular absorption. This tends to nullify the GLUT2-induced decrease in apical membrane net absorption.\n\nAdditional complexity is introduced by glucose-coupled Na+ and water flows altering cytosolic and interstitial osmolarities, thereby generating changes in enterocyte cytosolic and interstitial fluid volume and interstitial pressure. The interstitial pressure changes affect fluid and solute flows via the paracellular pathway and via the capillaries and lymphatics43. The effects on water flows are shown with green arrows in Figures 1A–D. As modelled here, changing the maximal rate of apical GLUT2 or capillary perfusion rates have relatively smaller effects on net water than on glucose flows.\n\nThis is also illustrated in Figures 2A and B, where increasing apical GLUT2 activity from zero (Figure 2A) to a high level (Figure 2B), increases GLUT2 backflux and also enhances glucose influx via the paracellular route. Consequently the net effect of altering apical GLUT2 activity on luminal glucose absorption is almost zero.\n\nIncreased paracellular glucose diffusion has multiple effects on glucose fluxes and accumulation. Increasing paracellular glucose permeability directly increases paracellular glucose flux (Figure 3A (blue)). This increases the interstitial glucose concentration (Figure 3C (blue)). Raising interstitial glucose concentration decreases the glucose concentration gradient across the basolateral membrane, thereby decreasing basolateral glucose flux and raising cytosolic glucose concentration (Figure 3C (red)). Increasing cytosolic glucose concentration reverses the direction of glucose flow across the apical membrane via GLUT2 (Figure 3B (red)), but is without significant effect on glucose flux via SGLT1 (Figure 3A), or cell volume (Figure 3B (blue)).\n\nThus, it is evident that paracellular glucose diffusion significantly alters glucose fluxes, both directly via the paracellular and indirectly on the passive glucose fluxes at the apical and baso-lateral membranes. Only when paracellular glucose flux is close to zero is there any significant glucose influx via GLUT2 (Figure 3B).\n\nThe effect of phloridzin is to block SGLT1 without affecting GLUT21. As previously discussed, phloretin inhibits GLUTs 1-IV, but not GLUT5. However, it has additional effects on chloride, urea and water permeability, so also affects paracellular conductivity22,23.\n\nSimulation of the temporal effects of phloridzin on intestinal glucose uptake exposed to luminal glucose 30 mM1 shows that whilst inhibiting glucose influx via SGLT1, net glucose efflux via GLUT2 is abolished as a result of the decreased uphill glucose accumulation in the cytosol. Hence glucose flux across the basolateral membrane is reduced; however, because the interstitial glucose decreases due to diminished, transcellular flow paracellular glucose influx rises. Consequently, the net effect of SGLT1 inhibition by phloridzin on net glucose absorption is negligible, as observed in rabbit ileum pre-incubated with glucose9. Following phloridzin inhibition of SGLT1, cytosolic glucose falls from ≈ 32 mM to ≈ 17 mM as simulated here (Figure 4A and 4B).\n\nGLUT2 is present in the apical membrane Vmax 2 and the capillary perfusion rate = 10 is similar to that shown in Figure 2 panels B and D. The luminal glucose is 30 mM and afferent capillary glucose is 5 mM to simulate the conditions used by Kellett & Helliwell 20001. In Figure 4, panels A and B inhibition of SGLT1 activity at 0.5h to zero reduces glucose flux via SGLT1 to zero. Simultaneously glucose flux via GLUT2 increases thereby reversing the backflux from -1.1. to 0.05 and also paracellular glucose flux increases from 0.55 to 0.95. In panels C and D the glucose concentration changes in the cytosol (red) interstitial fluid (blue) capillary fluid (black) and cytosolic volume (green). Following phloridzin addition and inhibition of SGLT1, cytosolic glucose falls from 33 to 30 mM; cytosolic volume falls from 0.62 to 0.56 ml at 1 hour without significant changes in capillary or interstitial fluid glucose concentration. This explains both the fall in glucose reflux via GLUT2 and the decrease in basolateral membrane flux is compensated by the rise in paracellular flux thereby nullifying interstitial glucose concentration changes.\n\nPhloretin addition at 1h is simulated by blocking apical GLUT2 (panel A) and by blocking both apical GLUT2 and paracellular glucose and Na permeability (panel B). GLUT2 fluxes fall to zero in both panels A and B and in panel A there is a small increase in paracellular glucose flow but the total transluminal glucose flux is unaffected by addition of phloretin after phloridzin. There is a small decrease in cytosolic volume from 0.56 to ≈ 0.55. the paracellular flux falls to zero as does the transluminal glucose flux in panel B, simulating the effect observed by Kellett and Helliwell 20001. This is accompanied by a large decrease in cell volume from 0.56 to 0.51 ml. Since no net glucose transport now occurs from the luminal fluid, capillary glucose concentration also decreases to 5 mM.\n\nSubsequent inhibition of apical GLUT2 by phloretin is accompanied only by a very small decrease in net glucose influx as it falls to zero. However, this decrease in net transcellular glucose influx is supplemented by a reciprocal increase in paracellular flux, so that there is still a negligible change in net luminal glucose absorption. Thus, when glucose fluxes via apical SGLT1 and GLUT2 are completely inhibited, only the paracellular route remains to permit luminal to submucosal glucose flow and this flux rises to compensate for the reduced transcellular flow as a result of reduced interstitial glucose concentration.\n\nThe simulation shows that if phloretin inhibits only apical GLUT2, then it exerts no significant effect on luminal glucose uptake Figure 4A. If instead of only inhibiting glucose flux via apical GLUT2, phloretin also inhibits paracellular glucose and electrolyte fluxes, then the observed effect on intestinal glucose absorption (Figure 4B) is similar to that observed by Kellett and Helliwell1; namely, reduction in net luminal glucose flux to zero. The cytosolic glucose together with the interstitial glucose concentrations now fall to 5 mM; equal to the sink capillary glucose concentration, since now there is no compensatory rise in paracellular glucose flux occurring when interstitial glucose concentration is reduced.\n\nGLUT2 functions as an apical glucose shunt, thereby reducing cytosolic glucose accumulation by SGLT1. This shunt functions in two important ways, first by reducing net luminal influx, rather than increasing it as previously deduced1. It will also redistribute the luminal glucose to more distal intestinal regions, consequently exposing larger intestinal surface areas to luminal glucose.\n\nThis latter effect may explain why when pigs are exposed to high carbohydrate diets raised SGLT1 protein and mRNA expression is observed in more distal intestinal regions44. More SGLT1 is also observed in duodenal epithelia of morbidly obese humans45. Increased density and increased area of intestinal SGLT1 expression implies that the intestine develops the capacity to deal with increased carbohydrate loads by absorbing more carbohydrate in aggregate, although not per unit area (Figure 5). This will generate higher concentration peaks of carbohydrate in the splanchnic circulation following absorbable carbohydrate ingestion41,45 and higher rates of splanchnic blood flow in conscious animals46–48.\n\nThe KO cells have higher enterocyte glucose concentrations in the proximal intestine, but higher paracellular flow and larger cell volumes. The normal enterocytes have lower cytosolic glucose concentrations and SGLT1 is more widely dispersed along the intestinal length with higher rates of glucose permeation in distal regions of the small intestine. Long term exposure may lead to higher maximal glucose absorption rates in normal intestine than with GLUT2 KO.\n\nIt would seem more likely that instead of a means of enhancing apical glucose absorption, GLUT2 behaves primarily as an osmoregulator to maintain enterocyte volume in the face of large and rapid changes in the luminal and cytosolic osmotic pressure following ingestion of carbohydrates, or their subsequent dilution upon drinking water.\n\nSince glucose is one of the most variable osmolytes within the intestine and splanchnic circulation, it is likely that a rapid adaptation to hyper or hypo osmotic changes within the intestinal lumen via a GLUT2 shunt pathway in the apical membrane would provide a useful means of regulating enterocyte volume, thereby avoiding excessive membrane stress and cytolysis. The reduced local net influx would also result in redistribution of hypertonic luminal glucose to more distal regions where this excess glucose would be absorbed by SGLT1.\n\nGLUT2 has not previously been considered as an osmoregulator of enterocyte volume. This role has been mainly assigned to potassium and chloride channels49. Whilst ion channels certainly provide an important role in cell volume regulation, they may not be as well adapted as GLUT2 to fulfilling the enterocytes’ specialized needs for osmotic control due to large changes in sugar dependent osmotic gradients.\n\nSimulation shows that glucose accumulation within the cytosol via SGLT1 is accompanied by an increased cytosolic volume Figures 2C and 2D, Figures 4C and 4D. The effect of increased rates of apical GLUT2 which prevents excessive glucose accumulation at high luminal glucose concentrations, compare Figure 2C with Figure 2D, also reduces enterocyte volume increase.\n\n\nSummary and conclusions\n\nKellett & Helliwell (2000)1 have proposed that the non-saturable component of intestinal glucose absorption, apparent when luminal glucose is raised above 15 mM, is due to enhanced flux via the low affinity glucose transporter GLUT2, which they and others have observed32 is present within the apical border of the jejunum and ileum following prolonged exposure to high intraluminal glucose or following activation of protein kinase C by phorbol myristate acetate.\n\nEvidence in support of this contention is that this “non-saturable” component is inhibited by high phloretin or high cytochalasin B concentrations – which both can inhibit GLUT2. The Km of the phloretin sensitive component claimed to be similar to that of GLUT2 approximately 56±14; n = 1.6±0.4.\n\nThey argue that GLUT2 is the most likely route for this low affinity transport, since it also transports fructose and the only other fructose transporter GLUT5 is insensitive to inhibition by either cytochalasin B or phloretin.\n\nHowever, it is unclear that upregulation of GLUT2 within the intestinal brush border actually does enhance D-glucose absorption. At raised luminal glucose concentrations the cytosolic concentrations and the submucosal interstitial fluid glucose concentrations will exceed the intestinal luminal glucose concentrations, so GLUT2 will stimulate passive downhill glucose reflux from the enterocyte cytosol, thus reducing net glucose uptake across the luminal surface. This glucose backflux may be augmented by glucose reflux via the paracellular pathway when the interstitial glucose is raised. This will occur when the splanchnic capillary glucose concentration is raised above 10 mM, as occurs during ingestion of high glucose loads, or in hyperglycaemic states.\n\nThese states are simulated here with a model of intestinal glucose transport incorporating glucose sodium and water cotransport across the luminal border variable rates of apical GLUT2 and paracellular flows and variable rates of capillary clearance of solutes and water from the submucosal interstitial fluid.\n\nThe model demonstrates that apical membrane GLUT2 may usefully function as osmoregulator to prevent excessive enterocyte volume changes during glucose loading, or following sudden decreases in luminal glucose concentration.\n\n\nMethods\n\nThe simultaneous flows of glucose Na and water from lumen across the apical membrane to cytosol and across the intercellular junctions from lumen to interstitial space followed by flows across the basolateral membrane of glucose Na and water to the interstitial space and from the interstitial space to the capillary lumen are modelled using Berkeley Madonna version 9.0119 http://www.berkeleymadonna.com/. Water flows generated by the osmotic pressure generated across the membrane boundaries between adjacent compartments generate volume changes in the cytosol and interstitial compartments. These volume changes are controlled by independent apical and baso-lateral hydraulic coefficients. Additionally, Na+ and glucose flow via SGLT1 and GLUT2 are assumed to generate a coupled water flow50,51 and modelled as an additional component of water flux across both apical and basolateral membranes. The interstitial fluid compartment is assumed to have a non-linear elasticity similar to that observed by Granger52 so that interstitial pressure rises non-linearly with volume.\n\nThe Na glucose and coupled flows and uncoupled flows via SGLT1 are modelled as outlined in53 glucose flow across both apical and basolateral GLUT2 is modelled according to a simple two site model\n\ni.e Jglucose = (Gout/(1+Gout)-Gin/(1+Gin).Vmax;\n\nwhere Gout and Gin are the glucose concentration/Km(GLUT2) in the adjoining membrane compartments and Km GLUT2 is the assigned GLUT2 Km = 17 mM. Na flux basal-lateral membranes is assumed to have a similar kinetics between two to saturable sites to that of glucose Km Na = 25 mM.\n\nAdditionally Na is pumped from the cytosol into the interstitial solution, according to the simple saturation equation\n\nJNapump = Nacyt.Vm(Napump)/(Nacyt+Km(Napump)).\n\nFlows of glucose and Na between the interstitial fluid and capillary fluid are assume to take the form of the convective diffusion equation\n\nJi = Jw(Co+Ci)/2 + Pi(Co-Ci).\n\nWhere Ji is the solute flux and Jw is water flux between interstitial fluid and capillary fluid Co and Ci are the external and internal concentrations of solutei and Pi is the permeability of solutei.\n\nThe water flow across the paracellular pathway Jwcpc is determined by the osmotic and hydraulic pressure difference between the luminal and interstitial solutions hence Jwcpc = Lp (2(Nain-Nalum).σNa + ((Gin-Glum).σGlc - Pin) where iin and ilum are the osmotic pressure of solutes I in the luminal and interstitial fluids σi is the reflection coefficient of solute I and Pin the interstitial pressure mm Hg.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAppendix {comments are italicised with curly brackets}\n\nd/dt (Cytgl)= + Japgl – JGlbl { Cytgl = cytosolic glucose mM Japgl = glucose flux across the apical membrane = SGLT1+ GLUT2 flux; JGlbl = basolateral glucose flux.}\n\nINIT Cytgl = 0 /Cytvol { INIT = initial ; cytvol = cytosolic volume /cm2 }\n\nLIMIT Cytgl >= 0\n\nd/dt (CytN)} = + JNap – JNbl {CytN = cytosol Na mM; JNap = Na flux across apical membrane via SGLT1 coupled and uncoupled; JNbl = Na flux across baso-lateral membrane = Na pump flux and passive Na flux}.\n\nINIT CytN = 1.5/Cytvol\n\nLIMIT CytN >= 0\n\nd/dt (inGlc) = + Jglpc - JcapGl + JGlbl { Jglpc = paracellular glucose flux; JcapGL= glucose flux from interstitial to capillary fluid;}\n\nINIT inGlc = 0/Invol {inGlc = interstitial glucose concentration}\n\nd/dt(InNc) = - JNcap + JNbl + JNpc { JNcap {inNc= interstitial Na concentration; JNcap = Naflux from interstitial to capillary fluid; JNbl Na flux across basolateral membrane}\n\nINIT InNc = 0/{Invol}\n\n\n\nLIMIT InNc >= 0\n\nd/dt(Invol)= + Jblw - Jcapw + Jpcw {Invol = interstitial volume; Jblw = water flux across basolateral membrane; Jcapw= water flux between interstitial fluid and capillary fluid; Jpcw = paracellular water flux}\n\nINIT Invol = 0.1 {invol= interstitial volume ml}\n\nLIMIT Invol >= 0\n\nd/dt (Cytvol) = - Jblw + Japw {Cytvol = cytosolic water volume; Jblw water flow across basolateral membrane includes osmotic and sugar coupled flows; Japw= waterflow across apical membrane includes osmotic and sugar coupled flows.}\n\nINIT Cytvol = 0.5\n\nLIMIT Cytvol >= 0\n\n\nReferences\n\nKellett GL, Helliwell PA: The diffusive component of intestinal glucose absorption is mediated by the glucose-induced recruitment of GLUT2 to the brush-border membrane. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "7370", "date": "19 Jan 2015", "name": "George Kellett", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBACKGROUNDNumerous papers over eight decades since 1935 have reported that intestinal glucose absorption comprises two components, one secondary active and mediated by SGLT1, the other a highly regulated “diffusive” component occurring predominantly at concentrations well above those required to saturate SGLT1. In 1987, Pappenheimer proposed that the “diffusive” component was mediated by SGLT1-dependent solvent-induced paracellular flow through tight junctions, as a result of the concentration of glucose up to ~300 mM in the intercellular spaces2. The field was split by the ensuing debate in which Diamond and Ferraris contested the concept of paracellular flow3-6. In so doing, they implied that SGLT1 is the only pathway of glucose absorption and denied the existence of two components. Subsequently, Kellett’s (my) laboratory proposed that the “diffusive” component is mediated by the glucose- and SGLT1-dependent transient insertion of the low affinity basolateral transporter GLUT2 into the apical membrane at high glucose concentrations, so that GLUT2 then mediated a major pathway of transcellular glucose absorption7-9. Detailed mechanisms for the regulation of apical GLUT2 insertion by diet, sugars, peptides, Ca2+, artificial sweeteners, hormones and obesity have been described10-18. In effect, the apical GLUT2 model replaced paracellular flow and the impasse in the long-standing debate seemed resolved. Nevertheless, as cited, some workers have recently reported difficulty detecting apical GLUT2. They therefore continued to deny the existence of any significant pathway other than SGLT1. In fact, the known properties of apical GLUT2 dietary regulation explain both cases. In the report by Gorboulev et al.19,20 mice were starved, which reduces apical GLUT2 and enhances SGLT1; mice also have higher SGLT1 activities than rat. In the report by Roder et al. 21 using SGLT1 knockout mice, KO and wild-type animals were necessarily maintained on sugar-free diets, which are well known to diminish apical GLUT2 greatly.17 A NOVEL PARACELLULAR FLOW PROPOSALProfessor Naftalin has undertaken the truly daunting task of seeking to provide a comprehensive, integrated model of intestinal glucose absorption. He accepts there are two components of glucose absorption and also that GLUT2 is transiently inserted into the apical membrane. However, in a further twist to the debate, he seeks to reinstate a central role for paracellular flow and thereby reinterpret that of apical GLUT2. In essence, it is proposed that glucose transport through SGLT1 results in accumulation of cytosolic glucose to supraluminal concentrations in enterocytes. At that point, net absorption of glucose through apical GLUT2 switches to net secretion of accumulated glucose through apical GLUT2 into the lumen; it is then transported across the epithelium by a paracellular channel, along with other luminal glucose. Apical GLUT2 therefore acts, not as a direct transcellular transporter, but as a shunt to supply a major paracellular pathway of transepithelial absorption. Paracellular flowThe proposal depends absolutely on the view that paracellular flow of glucose through tight junctions exists and, furthermore, that it occurs with rates comparable to observed luminal disappearance in perfusions in vivo. However, the concept of paracellular flow advanced by Pappenheimer lost favour during the debate with Diamond and Ferraris22. In particular, they argued convincingly that paracellular markers, if they cross the intestinal barrier at all, do so only in low amounts after experiments of many hours or even longer, when the integrity of tissue becomes questionable. Even in cited example of cat, reported paracellular flow of glucose is only ~10% of glucose absorption. Thus the rate of absorption of paracellular markers is minimal to negligible compared with that for glucose. Nevertheless, because of its potential importance, my laboratory made strenuous attempts to detect paracellular flow12. Mannitol, the “gold standard” of paracellular markers was used, since L-glucose has a low affinity for SGLT1. Rats were maintained on a high carbohydrate diet and food was flushed from jejunum just before perfusion in vivo; the perfusion was single pass at low flow rate and net glucose absorption (total absorption minus secretion) was determined chemically as luminal disappearance. At 75 mM luminal glucose, the steady-state rate of glucose absorption was established within just 15 min and was some 200-fold greater than for mannitol; see Table 1 of ref 12. “Closing” tight junctions by inhibiting myosin phosphorylation had no effect on mannitol clearance; nor did clearance change over a wide range of glucose concentrations at constant osmolarity (balanced with mannitol to 75 mM total sugar). Changes in water absorption correlated with changes in rapidly inserted apical GLUT2, not mannitol clearance. Studies from other groups lead to same conclusion. In the work of Brot-Laroche and colleagues on apical GLUT2, fructose transport studies were done in vesicles17; paracellular flow could not be involved. Apical GLUT2 mediated 60% of fructose transport, the rest was by GLUT5. The results with GLUT2 knockout mice correlated well with fructose and glucose perfusion data, one could be predicted from the other. Since GLUT5 and GLUT2 are facilitative transporters, there can be no concentration of the transcellular gradient in vivo, yet fructose absorption at high luminal concentrations in vivo shows the same apparent linearity as glucose2. Resistin23 and metformin24 promote AMP-mediated rapid insertion of apical GLUT2 in mice and rat to result in an increase in associated glucose absorption; there is no effect on minimal mannitol clearance. Notably, SGLT1 membrane density and ∆Isc were halved. This switch away from SGLT1 at high glucose concentrations was first observed in a study with the powerful AMP-kinase activator, AICAR, which induces a 3-fold increase in glucose absorption through a comparable increase in apical GLUT225. Simultaneously, SGLT1 is degraded to such an extent as to be almost undetectable (within ~30 min), so that only GLUT2 is at the apical membrane. The switch to the facilitative, energy-independent apical GLUT2 may represent a response to energy stress as the energy-dependent SGLT1 reaches maximal capacity. Reports that paracellular flow is minimal to negligible under the conditions of apical GLUT2 studies have not been cited. Nor have any new experimental data been presented to change the debate on paracellular flow. The integrity of the intestinal barrier is paramount, as immunologists surely agree. Intestinal transport is simulated with a computer-based modelThe novel paracellular flow proposal is based entirely on computer simulation of what is described as a “multiplicity and complexity of interactive processes”. As the extensive mathematical appendix so lucidly shows, simulation depends on many steps and their interactions, the choice of many parameters and their values, the concentrations of glucose and Na+ at specific places within various compartments, and so on. Inevitably, a serious issue is that some, possibly most, values are of great uncertainty or simply unknown. Two key examples suffice to make the point. The Kt of glucose for SGLT1 is taken as 17 mM, in contrast to reports of 2326 and 26 mM7 in vivo. The Kt of GLUT2 is also taken as 17 mM, reported for expression in oocytes. In contrast, the value of 56 ± 14 mM, based on an empirical sigmoid curve analysis of in vivo data7, is described as “very high”, implying the “diffusive” component is not GLUT2 but paracellular flow. In fact, the Kt of GLUT2 for uptake is 48 ± 5 mM for basolateral membrane vesicle preparations27. Moreover, the question of asymmetry in this bidirectional transporter seems not to have been addressed: Vmax for uptake is ~6-fold greater than for efflux27. Such differences and uncertainties for most parameters surely have a considerable impact on simulation. Indeed, simply taking the Kt values of SGLT1 and GLUT2 to be the same as each other and much lower than the literature consensus strongly biases simulation outcome. Transcellular Glucose Gradients In VivoIt is widely accepted that secondary active transport by SGLT1 results in glucose accumulation to enterocyte concentrations greater than in the lumen of intestinal preparations in vitro. This is because the Kt for SGLT1 is very much lower than in vivo (sub mM v 26 mM) and because preparations such as everted sacs have poor clearance. The question is whether glucose accumulation occurs in vivo. If, as the balance of evidence indicates, there is negligible paracellular flow, then the transcellular gradient must be downhill (l-m-s) when luminal concentrations are high. Other evidence supporting this conclusion has been reviewed3-5. Recycling of Glucose and Na+ Across IntestineRecycling of certain key nutrients across the intestine is vital to its function. Thus Na+ is recycled paracellularly to the lumen at high rates through claudin-15, to support continued activity of SGLT1 and other Na+-dependent transporters28. Knockout of claudin-15 results in abnormally low luminal Na+ (~8 mM v ~57 mM for wild-type) and a large impairment in glucose absorption (∆Isc). Recycling of Na+ is important when luminal glucose concentrations are low, e.g. between meals or overnight, in starvation and desquamation. Thus SGLT1 is the only transporter capable of driving glucose uphill against its gradient to plasma and therefore preventing loss of glucose by secretion through apical GLUT2. Conversely, glucose is important for Na+ recovery. A clear example is streptozocin-diabetes, which is characterised by hyperglycaemia and hyponatraemia; a permanently high level of apical GLUT2 is also found in diabetic rats29. When luminal glucose is less than in plasma, secretion through apical GLUT2 16 is three-fold greater in diabetic than in normal rats and is increased by phloridzin 30. Thus recycling of glucose through apical GLUT2 is associated with recovery of Na+ through SGLT1, providing potential compensation for loss of Na+ by urinary excretion in diabetes. SGLT1 will also play a role in recovery of water secreted by apical GLUT2. In the scenarios just outlined, there is clear physiological advantage to secretion of either Na+ or glucose in their mutual recovery at low luminal concentrations. At high concentrations in the absence of paracellular flow, the switch to facilitated transcellular absorption through apical GLUT2 would be energy efficient and, as noted for periods of energy stress, might even be accompanied by a reduction of energy-dependent SGLT1. All that is required is a natural reversal of the transcellular concentration gradient in response to a fresh luminal glucose load. Energy-linked recycling of metabolic substrates used to be thought of as a waste of energy, a so-called “futile cycle.” It is now recognised as an amplification mechanism for rapid mobilisation of fresh substrate, or, by analogy, uptake of incoming glucose when absorption and secretion are initially comparable at low luminal glucose concentrations. In addition to its roles as a transporter and in the mutual recovery of Na+ and glucose, SGLT1 plays a major role in regulating apical GLUT2. SUMMARYThis paper highlights the sheer complexity of developing an integrated model of intestinal glucose absorption. Such contributions to the literature are rare; they are valuable for forcing us to think about many things that we have yet to learn about, including apical GLUT2 and the in vivo situation, and to promote debate. Significant issues are inevitable, though, when such an ambitious objective is in mind. The novel model places paracellular flow at the heart of intestinal glucose absorption. However, new experimental evidence necessary to justify such a role is not presented. The account of the debate on paracellular flow is very selective and gives the erroneous impression that the concept is widely accepted. At best, it is controversial. A fuller account should be given. Specifically, recent papers from several groups report that paracellular flow is minimal under the conditions of apical GLUT2 studies, when large changes in absorption, apical GLUT2 and even SGLT1 are seen. Reinterpretation of apical GLUT2 data to support the novel paracellular flow model is not possible. Simulation results are presented for just one luminal glucose concentration (50 mM) and two rather selective values of Km for key transporters; there is no consideration of GLUT2 asymmetry. A much wider range of factors, concentrations and parameter values should be explored, including the absence of paracellular flow. There is significant literature evidence to support the view that, when the glucose concentration in the lumen is much higher than in plasma, the transcellular glucose gradient (l-m-s) is downhill all the way in vivo. The differences between in vivo and in vitro situations should be clarified. Emphasis on recycling of glucose through apical GLUT2 has been as a shunt to serve paracellular flow. Evidence that recycling of glucose and Na+ through SGLT1 is vital to intestinal function should be included. New title? “Computer simulation of an integrative model of intestinal glucose absorption”", "responses": [ { "c_id": "1191", "date": "22 Jan 2015", "name": "Richard J Naftalin", "role": "Author Response F1000Research Advisory Board Member", "response": "Dear Professor Kellett (George, GK),Thank you for reviewing my commentary on the role of apical GLUT2 in intestinal absorption of glucose, whose main focus is examining quantitatively how apical GLUT2 interactions affect intestinal glucose transport. Your research has generated much interest and controversy over the last fifteen years. I am fairly convinced that the problem that it has resurrected – namely, coping strategies with excessive glucose absorption and its subsequent accumulation within the splanchnic circulation, has important implications for the pathophysiology of glucose absorption and metabolism. The spin offs implicating the role of gut incretins, autocoids and hormones in control of glucose appetite, taste and absorption with ramifications in such topics as obesity and type II diabetes are likely to be of critical importance and subject of research for the foreseeable future. Nevertheless, I am in substantial disagreement with some of the topics you raise. Some of the points I will make refer to simple clarifications and others are more fundamental. Point 1: You GK say “At that point, net absorption of glucose through apical GLUT2 switches to net secretion of accumulated glucose through apical GLUT2 into the lumen; it is then transported across the epithelium by a paracellular channel, along with other luminal glucose. Apical GLUT2 therefore acts, not as a direct transcellular transporter, but as a shunt to supply a major paracellular pathway of transepithelial absorption”. Possibly, there is a slight misreading here. GLUT2 does indeed function as an apical membrane shunt facilitating glucose reflux, thereby reducing cytosolic glucose concentration. This has the secondary effect of reducing net glucose flux across the baso-lateral membrane, thus reducing interstitial glucose concentration and thereby increasing the concentration gradient between the lumen and interstitial fluids. This increased concentration gradient generates the increased diffusive flux via the parallel paracellular pathway. No adjustment of paracellular parameters is required to accommodate this increased paracellular flow. The mere fact that a paracellular route is present in parallel with the transcellular route will generate such an increase in flow if transcellular flux is reduced inhibited. Point 2: GK contends that the (Ferraris & Diamond 1997) review eliminates the possibility of a significant functional paracellular route for glucose absorption - as you say “Diamond and Ferraris contested the concept of paracellular flow3-6….. In so doing, they implied that SGLT1 is the only pathway of glucose absorption and denied the existence of two components” and later you say “the apical GLUT2 model replaced paracellular flow and the impasse in the long-standing debate seemed resolved”. Perhaps! But, then again maybe not – nothing much in Physiology is wholly resolved. The implication is that Ferraris and Diamond, refuted the functional existence of the paracellular absorptive pathway is contradicted by substantial literature in which the in vivo technique of monitoring passive intestinal permeability using dual sugar absorption technique e.g. lactulose and rhamnose (Laker & Menzies 1977; Bjarnason et al. 1995) demonstrates a considerable element of passive intestinal absorption of un-metabolized sugars in humans in vivo. Furthermore, it is evident that neither Diamond nor his erstwhile colleagues, Ferraris and Karasov, any longer hold fast to the view that paracellular sugar fluxes are a negligible component of intestinal absorption. Passive paracellular glucose absorption is now thought to be the dominant route of absorption in nectarivores and be of major importance in several reptiles, rodents and mammals. It is apparent that there is wide species variability in passive intestinal absorption of sugars: from highest in the American robin with 92% passive (paracellular) absorption to lowest, 2.1 % in rabbits (Karasov & Cork 1994; Secor et al. 1994; Ferraris 2001). Humans have around 20% passive absorption, assuming they have not been drinking alcohol excessively, or not treated with non-steroidal anti-inflammatory drugs, when the paracellular flow increases by at least two-fold (Bjarnason et al. 1984). Thus, I do not agree with your assertion that paracellular glucose transport is generally negligible – this is particularly not the case when luminal glucose concentrations exceed 25mM. The low mannitol “clearance” you refer to should be compared with the passive, rather than the active component of glucose absorption. Passive glucose and galactose loss from rat jejunum in the presence of 0.5mM phloridzin does not differ significantly from sorbose - which is poorly selective for GLUTs and SGLTs (Debnam & Levin 1975). Point 3: A clarification: the assigned Kt of glucose for SGLT1 transport is 2mM and for Na is 25mM and for GLUT2 at both apical and basolateral membranes the Kt is 17mM. This requires clarification and I will amend the appendix and include these values in the legends to Figure 1. These values give an “operational” Kt for net glucose flux via SGLT1, as obtained from the model ≈17mM Figure 3A. The flux via apical GLUT2 with an absent paracellular component Figure 3B has an “operational” Kt around 25mM. These values are well within the observed values reported. For those unfamiliar with modelling complex processes, the operational Kt’s are not necessarily the same as parameters assigned to the transporter. The operational Kt for net transport is a lumped parameter that depends upon a large number of interactions. Operational “parameters” –are actually variables and only equal to the assigned parameters in the ideal zero-trans condition i.e. when the opposing side of the transporter contains zero ligand and back flux is zero. Whilst this condition may hold in a few in vitro conditions, such as uptake into oocytes, or exit fluxes from cells or vesicles into large volume of bathing solution, it is unattainable with net intestinal transport, where the cytosolic and interstitial fluids contain transported ligands at substantial concentrations.Using pharmacological tools in complex whole tissue preparations to dissect the parameters of individual transport process is likely to be a crude and sometimes misleading practice. However, adoption of more precise reductive approaches does not circumvent the problem of assessing how individual elements function within the integrated whole organ as is being examined here. This is the role of modelling and simulation which depends of course on use of experimental data. Apropos I am disappointed that GK has made no comment on the action of phloretin as he attributed to it the unique effect of inhibiting apical GLUT2(Kellett & Helliwell 2000). In addition to the other reported effects of phloretin I have cited (Wright et al. 2011) have mentioned that phloretin also inhibits SGLT1 (Ki 50 µM). They infer that Kellett and Helliwell underestimated the role of SGLT1 in intestinal transport. Point 4: GK contends that the simulation results are presented for just one luminal glucose concentration (50 mM) and two rather selective values of Km for key transporters; there is no consideration of GLUT2 asymmetry. A much wider range of factors, concentrations and parameter values should be explored, including the absence of paracellular flow. It is incorrect to imply that the results are presented at just one luminal glucose concentration. This is true only for figure 1. In figures 2 and 3, luminal glucose concentration is varied between 0 and 50 mM and the steady state transepithelial glucose transport rates are simulated in different conditions (see figure 3A and 4A). In figure 4, the luminal glucose concentration is held at 30mM to simulate the conditions observed by (Kellett & Helliwell 2000). With phloridzin present it is certainly correct that the luminal> cytosolic > interstitial glucose concentration. However when phloretin is added in addition to simulate the Kellett-Helliwell experiment and it is assumed only to inhibit apical GLUT2, no significant change in transepithelial glucose flux is observed. The reason for this is that the reduced transcellular flow is compensated by enhanced paracellular flow. Only when phloretin is assumed to block paracellular flow in addition to transcellular flow is any inhibitory effect of phloretin on transepithelial glucose transport observed. Many other conditions could be simulated e.g. varying Na concentrations, varying osmolarity etc; however the main point of this review is to illustrate some of the fallacies relating to apical GLUT2 function without excessive overload. Point 5 GK: There is no consideration of GLUT2 asymmetry… (Maenz & Cheeseman 1987) reported that the kinetic parameters observed in baso-lateral membrane vesicles from rat small intestine. They found that the Kt for uptake was 48±5 mM glucose, whereas for influx it was 23±2mM. A later review reported that GLUT2 in isolated hepatocytes has a Km of approximately 20mM and is symmetrical (Thorens 1993). I will add a reference to Thorens review on GLUT2. Point 6. GK makes the point that “Emphasis on recycling of glucose through apical GLUT2 has been as a shunt to serve paracellular flow. Evidence that recycling of glucose and Na+ through SGLT1 is vital to intestinal function should be included.”I take this to mean glucose and Na recovery from the lumen via SGLT e.g. during periods of starvation e.g. at night. It is certainly likely that glucose may leak via the transcellular route when the lumen is glucose free. This may also occur via the paracellular route and recovery via SGLT is an important function. This point was made very well by (Boyd & Parsons 1976) and says much about SGLT capacity to recapture glucose leaked via either trans or paracellular routes from the blood but no much about the specific role of apical GLUT2. However it is not very relevant to the main topic of debate here, which is the mode of intestinal transport with high luminal glucose concentrations. Point 7. GK suggests a new title “Computer simulation of an integrative model of intestinal glucose absorption” I do not feel that a changed title is a warranted. I agree that computer simulation of net glucose transport lies at the heart of this commentary, however this is used simply as a tool- a means to illustrate the cardinal point that GLUT2 does not have a functional role in augmenting net glucose transport in the way that GK has suggested - namely during luminal overload. However by its possible alternative roles in spreading the luminal glucose load over a wider absorptive area and also preventing osmotic stress, GLUT2 induction may augment net glucose transport, as has been shown. ReferencesBjarnason, I., MacPherson, A. & Hollander, D., 1995. Intestinal permeability: An overview. Gastroenterology, 108, pp.1566–1581.Bjarnason, I., Peters, T.J. & Wise, R.J., 1984. The leaky gut of alcoholism: possible route of entry for toxic compounds. Lancet, 1(January), pp.179–182.Boyd, C. & Parsons, D., 1976. Effects of vascular perfusion on the accumulation distribution and transfer of 3-O-methyl-D-glucose within and across the small intestine. J Physiol, 274, pp.17–36.Debnam, B.Y.E.S. & Levin, R.J., 1975. AN EXPERIMENTAL METHOD OF IDENTIFYING AND QUANTIFYING THE ACTIVE TRANSFER ELECTROGENIC COMPONENT FROM THE DIFFUSIVE COMPONENT DURING SUGAR ABSORPTION MEASURED IN VIVO From the Department of Physiology , University of Sheffield , side have been measured in. J Physiol, pp.181–196.Ferraris, R. & Diamond, J., 1997. Regulation of intestinal sugar transport. Physiological Reviews, 77(1), pp.257–302. Available at: http://physrev.physiology.org/content/77/1/257.short.Ferraris, R.P., 2001. Dietary and developmental regulation of intestinal sugar transport. The Biochemical journal, 360, pp.265–276.Karasov, W.H. & Cork, S.J., 1994. Glucose absorption by a nectarivorous bird: the passive pathway is paramount. The American journal of physiology, 267, pp.G18–G26.Kellett, G.L. & Helliwell, P.A., 2000. glucose-induced recruitment of GLUT2 to the brush-border membrane. Biochem J, 162, pp.155–162.Laker, M. & Menzies, I., 1977. Increase in human intestinal permeability following ingestion of hypertonic solutions. J Physiol, 265, pp.881–894.Maenz, D.D. & Cheeseman, C.I., 1987. The Na+-independent d-glucose transporter in the enterocyte basolateral membrane: Orientation and cytochalasin B binding characteristics. The Journal of Membrane Biology, 97, pp.259–266.Secor, S.M., Stein, E.D. & Diamond, J., 1994. Rapid upregulation of snake intestine in response to feeding: a new model of intestinal adaptation. The American journal of physiology, 266, pp.G695–G705.Thorens, B., 1993. Facilitated Glucose Transporters in epithelial cells. Annu Review Physiol., pp.591–608.Wright, E.M., Loo, D.D.F. & Hirayama, B.A., 2011. Biology of human sodium glucose transporters. Physiological reviews, 91(2), pp.733–794. Available at: http://www.ncbi.nlm.nih.gov/pubmed/21527736 [Accessed September 10, 2014]." } ] }, { "id": "7045", "date": "20 Jan 2015", "name": "Hannelore Daniel", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper by R. Naftalin addresses a facet of gastrointestinal physiology that has for decades caused highly controversial discussions amongst the experts. Carbohydrates in our diet represent the quantitatively most important class of nutrients and a large fraction of our daily caloric intake. After digestion in the upper small intestine glucose derived from starch, glycogen, sucrose and lactose is the nutrient with the highest absorption rate of all dietary components and yet, we still do not understand in details how this is accomplished. There is no doubt that the rheogenic SGLT1 transporter in the brush border membrane is of prime importance and this is known from studies utilizing phloridzin that inhibits SGLT1 with high affinity, from humans suffering from hereditary glucose-galactose malabsorption and from mice lacking a functional SGLT1 protein. One of the critical issues is that SGLT1 when expressed heterologously in a target cell has a very high affinity whereas when glucose absorption is assessed in vivo either in rodents or in human intestinal perfusion studies affinities reported are one to two orders of magnitude lower. This discrepancy has been the starting point to search for additional glucose transporters that might be involved. Based on studies in rat intestine G. Kellett finally proposed that at very high luminal glucose concentrations SGLT1 transport function would induce a signalling cascade involving PKC that in turn would cause the recruitment of GLUT2 (a low affinity type uniporter) into the apical membrane from intracellular vesicles. This would allow glucose to move into the cell along a concentration gradient with GLUT2 simultaneously mediating also efflux across the basolateral side as the prime glucose transporting protein constitutively present in this membrane domain. However, as a uniporter GLUT2 could when incorporated into the apical membrane also cause a back-flux of glucose into the intestinal lumen in particular since SGLT1 allows uphill transport of glucose by its rheogenic transport mode. Since all biochemical studies published so far do not give conclusive and convincing data on whether GLUT2 can translocate into the apical membrane and thus contribute to overall glucose absorption, R. Naftalin now takes a modelling approach to assess how feasible a significant contribution of GLUT2 is. Since movement of bulk quantities of solutes (glucose and sodium) by the glucose transporters also induces osmotic gradients and in turn water movement, the conceptual work and the simulations presented also address water movement this as well as blood flow effects.\n\nA model has been build that is plausible and reveals the knowledge base of the author. This model allows changes in a variety of parameters to be visualized for the different compartments when glucose concentrations are increased. Simulations reveal that accumulation of glucose in the cytosol of intestinal epithelial cells mediated by SGLT1 is accompanied by increases in the cytosolic volume whereas an increased density of apical GLUT2 would prevent excessive glucose accumulation at high luminal glucose concentrations by back-flux that would also reduce the enterocyte volume increase. In this context GLUT2 fulfils primarily the role of an osmoregulator in controlling epithelial cell volume that is subject to rapid changes when bulk quantities of the solutes are absorbed. Although the work by R. Naftalin does not prove or disprove that GLUT2 has a important function in overall glucose absorption, it adds some novel ideas and other dimensions to the discussion. GLUT2 and its role in intestinal epithelial cells thus remains a mystery not only in its proposed function in the apical membrane but also for basolateral glucose efflux.", "responses": [] }, { "id": "7456", "date": "23 Jan 2015", "name": "Edith Brot-Laroche", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn his manuscript Pr R. Naftalin questions the role of apical GLUT2 in the regulation of intestinal sugar absorption. The manuscript comprises a view of earlier studies on absorption processes and proposes a theoretical model of glucose absorption in the small intestine. Since the main molecular features of sugar transporter in the small intestine have reached consensus, this timely manuscript opens a debate on the regulation of absorption processes including glucose transport, water flow and osmolarity issues. It is an ambitious project to describe a comprehensive model of intestinal sugar absorption considering the many different levels of regulation it involves. However in this manuscript, the kinetics of transporter expression in and out the enterocyte membranes in the control of absorption processes in sugar absorption were not taken into account in health and disease.The manuscript starts with a summary of 50 years of research effort in this area. In early days, glucose uptake in enterocytes comprised a Na-coupled electrogenic cotransport system mediated by SGLT1 clone by EM Wright 1, and a diffusive component first identified by its resistance to phloridzin inhibition or Na free conditions and later identified as a GLUT family members 2-4. The nature of an epithelial diffusive component was then debated and several mechanisms were proposed including paracellular flow and the presence of facilitated diffusion transport systems in the apical membrane i.e. GLUT5 for fructose and the low affinity transporter GLUT2 for glucose, galactose, fructose and mannose 2, 5. The exit of glucose from enterocytes was described to be mediated by GLUT2 in the basolateral membrane 5-7.More recent discoveries showed that GLUT2 can also traffic to the apical membrane of enterocytes in humans and rodents6, 7. GLUT2 trafficking in the apical membrane of epithelial cells has been demonstrated using various techniques including live imaging 8-10. Pr. Naftalin questions the relevance of apical GLUT2 in glucose versus water absorption. Major concernsPr Naftalin chose to focus on GLUT2-mediated osmoregulation but somehow neglected the dynamics control of sugar transporter densities in the apical and basolateral membranes of enterocytes. Although the mathematical modeling of this complex system is beyond my expertise, but may I suggest taking the time parameter as a key element in the control sugar absorption. Pr Naftalin reports that apical GLUT2 is not always seen, according to metabolic status of the mice or humans, an assertion that I can share with him. Indeed, apical GLUT2 is visible in animals consuming a sugar diet to increase GLUT2 expression and when glucose is abundant in the lumen. The transient presence of GLUT2 in apical membranes helps thus to understand how the intestine can absorb a large bolus of glucose that overcomes the absorption kinetic capacities of SGLT1. In pathologies altering insulin sensitivity, GLUT2 does not traffic anymore, remaining in apical membranes and increasing intestinal glucose absorption, worsening the status of subjects. In these conditions, apical GLUT2 can be readily observed. This rather simplifies the model of the transcellular transfer of glucose for apical membrane is permanently equipped with the GLUT2 transporter. The model should be amended to take these issues into account, in particular to include the varying levels of GLUT2 in the apical as well as basolateral membranes. This is also true for the SGLT1 transporter. Since transporters are also water channels 11-13 glucose transport will move water in the direction of the glucose gradient through the epithelial layer. Was that feature taken into account in the osmolarity model? Pr. Naftalin identifies conflicting data from my group on facilitative glucose transport, presumably by GLUT2, obtained using brush border membrane vesicles. I would like to give my interpretation of the data published at a time when transporters were characterized only by functional studies. My group identified a second transport system for glucose in addition to Na-cotransport using isolated brush border membrane vesicles, that is, in absence of interference of metabolism. The characteristics of this second system are very close to that identified later on for GLUT2 i.e. a glucose transport system with a low affinity (24 mM) and a high capacity 14. This transport is inhibited by cytochalasin B like GLUT2. In the cited papers there is no mention of phloretin inhibition. I suppose that the conflict also come from the nutritional manipulation of guinea pig15 comparing semi-starvation (25% of control food pair feeding experiment) to ad libitum feeding, we showed that the second transport functions was increased in absence of change in the contribution of the cotransport system. At that time, the physiological mechanism underlying the effect of semi-starvation was not analyzed further. Importantly, the effect of starvation was to increase the cotransport of glucose in absence of a contribution of system 2 as expected and confirmed in our studies in mice16. Therefore, it seems that are results are consistent over the different experimental analysis of sugar absorption and the role of GLUT2. The argument that “nonspecific transport of glucose at rates that are correlated with net fluid transport”17 is probably inappropriate since passive diffusion was measured in vivo through the urinary recovery of ingested sugars that constitutes an evaluation of passive permeability of the whole intestine including colon. These permeability parameters can hardly be applied as valid for the diffusive component ex vivo in selected part of the small intestine. Different experimental settings for absorption measurements probably cause the discrepancies in the evaluations of transporter Km for GLUT2 and SGLT1. Based on vesicle work 18 the Km for the sodium cotransporter and for the facilitative transporter in the BBM of the jejunum is in a ratio of 2 to 25 mM respectively. In addition, studies in rat intestine of Pr Kellet’s group, show cooperativity of the kinetics of glucose transport via GLUT2 6, 19. Figures in the model should take a better affinity ratio between SGLT1 and GLUT2 mediated transport activities. Minor point:GLUT5 is not a glucose transporter in intestine but a fructose transporter. Although it can transport glucose when expressed in Xenopus laevis oocytes. ConclusionThe title should include the words “model, mathematics” to stick to the content of the manuscript This manuscript describes the development of a mathematical model of sugar absorption in the intestine, a very challenging project indeed. The main merit is to put forward the many different levels of this complex and multi-compartment mechanism. The model is specially focusing on osmolarity issues in the absorptive process, this may be too far as rapid reading can trouble the reader understanding of the primary function of the transporters, which is primarily glucose transport. A model of this type should include the recent features of sugar absorption and include a kinetic description of events including the varying density of transporters in enterocyte plasma membranes. The role of GLUT2 in pathology should be described to show what are the enhanced and/or decreased elements of the model. Indeed, the absorption phase after a meal should be distinguished from the post absorptive time when the lumen is emptied of glucose. The relative affinities of transporters involved in transepithelial transport need to be corrected to stick to a ratio of affinities measured under the same experimental setting.", "responses": [ { "c_id": "1199", "date": "28 Jan 2015", "name": "Richard J Naftalin", "role": "Author Response F1000Research Advisory Board Member", "response": "Dear Prof. Brot Laroche (Edith, EBL)Thank you for your comments with regard to my commentary about the role of apical GLUT2 in intestinal glucose absorption. Your comment on the time scale of the changes raises an important issue which I did not fully consider in my initial treatment but clearly is worth addressing and I will do this now. The nice diagram (figure 5) in your paper (Ait-Omar et al. 2011) summarizes your view on the dynamics and routes of intestinal glucose absorption. The paracellular pathway is notably absent from this analysis. However as I have already mentioned in my answer to GK this is an important route in humans and has relevance to the question of the developing rates of intestinal glucose following acute luminal exposure to glucose during continuous infusion by duodenal gavage (Vanis et al. 2011). There are several components with widely differing equilibration rates relevant to quantitative description of luminal glucose absorption. First, the rate of delivery to the surface mucosa; this can be viewed as a rate of change of glucose concentration at a single point, or considered as an integral of the glucose content distributed over the entire absorptive surface. In considering the latter view, the more distal regions of small intestine will be exposed after the proximal and to a smaller overall load. This rarely considered point is treated with a simple approximation by Pappenheimer (Pappenheimer 1998). Obviously the rate of change of the integral glucose concentration along the entire length of small intestine will be much slower, than the rate as estimated at the proximal end. Link to figure S1 Following acute exposure to luminal glucose to 30mM, as modelled in Figure S1 A and B, luminal glucose concentration is simulated with an exponential rise (t½ ≈ 1-2min). This includes the mixing time within the lumen and diffusion through the unstirred layer to the enterocyte absorptive surface. Two other major rate processes determine net glucose absorption at the proximal end of the jejunum: the rate of glucose accumulation within the enterocyte cytosol; is difficult to measure in situ, but is assumed to be the main determinant of the rate of glucose absorption. The rate reported in isolated chicken intestinal epithelial cells (Kimmich 1968) has a t½ ≈ 1-2min. In vivo this rate likely to be faster as the force determining this rate is ultimately governed by Na-K ATPase activity which depends on the cell metabolic rate. The other secondary rates determining cytosolic glucose concentration are mainly via GLUT2, at both apical and baso-lateral membranes and the paracellular pathway. Kellett & Helliwell (2000) indicate that apical GLUT2 activity increases as a double exponential, a small faster component ( t½ ≈ 1-2min) and a larger slow component (t½ ≈ 77 min). I have replaced this double exponential with a single rate constant (t½ = 12 min) to describe the glucose stimulated increase in GLUT2 density at the apical membrane. The signal for increased apical is initiated when cytosolic glucose concentration >25mM. This occurs ≈ 1 min after exposure to 30mM luminal glucose. The simulation shows the effects of three steady state apical GLUT2 densities (0, 1 and 2 nominal units) (Figure 2 A and B). In the absence of apical GLUT2 glucose flux via GLUT2 is zero (red lines). As the density of GLUT2 increases, following an initial delay, the glucose flux becomes increasing more negative, reaching a steady state within ≈ 20 min. This increasing rate of glucose efflux is caused entirely by the slow rise in apical GLUT2 density. (Lower three lines Figure S1A). Also of note in this simulation are the transient changes in paracellular glucose flux. As apical GLUT2 density increases, paracellular glucose flux also increases. This, ; as previously noted (figures 2 and 4), is a consequence of the reduced transcellular glucose flux that increases the glucose concentration gradient between the lumen and submucosal interstitial fluid .The rate constants resulting from the luminal mixing and cytosolic accumulation almost entirely mask any observable glucose influx via GLUT2 occurring during the early phase of intestinal absorption, as envisaged by (Ait-Omar et al 2011) when the cytosol and submucosal glucose < luminal glucose. EBL “ Since transporters are also water channels 11-13 glucose transport will move water in the direction of the glucose gradient through the epithelial layer. Was that feature taken into account in the osmolarity model?” This question is extremely interesting- to me certainly -(see refs (50,51). It is evident that water is cotransported via a number of solute cotransporters particularly SGLT1 and GLUT2, and also NKCC(50). From other model simulations (not shown) it is apparent that water cotransport makes no substantial difference to the rates of net intestinal glucose or Na transport, but greatly affects glucose and Na-dependent water flows (not shown). Another noteworthy matter regarding water cotransport concerns the question as to how, when nectarivores imbibe more than three times their body weight of water per day with little salt content, they avoid becoming overhydrated. A solution appears to be that most of the water is retained within the intestinal lumen and is excreted via the cloaca, rather than kidneys (Karasov & Cork 1994; Caviedes-Vidal et al. 2007; McWhorter et al. 2013). A likely explanation for fluid retention within the nectarivore gut lumen appears to be that the paracellular glucose diffusion component is maintained at high levels by the by very high metabolic rates and very high rates of splanchnic perfusion that maintain the splanchnic capillary glucose concentration. Absence of water cotransport by glucose flow via the paracellular route is likely to be due to its very low reflection coefficient at the tight junction in these animals. Thus the high paracellular glucose absorption in hummingbirds also retards glucose absorption via the transcellular route; thereby preventing excessive water absorption by cotransport. EBL “The argument that “nonspecific transport of glucose at rates that are correlated with net fluid transport”17 is probably inappropriate since passive diffusion was measured in vivo through the urinary recovery of ingested sugars that constitutes an evaluation of passive permeability of the whole intestine including colon. These permeability parameters can hardly be applied as valid for the diffusive component ex vivo in selected part of the small intestine.” This is a question about which I have no expertise. However, it is probably inaccurate to say that the dual or multi sugar method does not give site-specific gastrointestinal permeability analysis, as has recently been shown (van Wijck et al. 2013). EBL “The relative affinities of transporters involved in transepithelial transport need to be corrected to stick to a ratio of affinities measured under the same experimental setting. ” In my answer to GK I explained that the assigned transporter parameters within the model relating to affinities are very similar to those obtained with isolated transporters tested in optimal conditions. The apparent operational affinities may differ greatly from those obtained in ideal conditions, as the concentrations of transported ligands cannot be held constant in the trans compartment(s) when the intestine is operating at near steady state absorption rates. EBL “The role of GLUT2 in pathology should be described to show what are the enhanced and/or decreased elements of the model. Indeed, the absorption phase after a meal should be distinguished from the post absorptive time when the lumen is emptied of glucose”. I would like to extend the model to various pathological situations, particularly T2D and hepatic cirrhosis and fatty degeneration and their relationships with obesity. This would require extension of the model to include hepatic glucose uptake and circulation (Alexander et al. 2001; Alexander et al. 2002; Li et al. 2003). However, it is evident from the simple (relatively) analysis already outlined that the local densities of transporters are not the sole factor determining the amplitude of glucose concentration and its flux within the splanchnic vascular bed. Another very important factor that has not been given enough attention is the extent of apical transporter distribution within the intestine and how this affects glucose absorption. If it is assumed that the distribution of SGLT1 and GLUT2 normally covers 30-40% of the available small intestine area ( 4200 cm -2) (Pappenheimer 1998; Soergel et al. 1968) i.e. 1000-1500 cm-2, then the maximal absorption rate for the entire intestine is approximately 1.0 mmole min-1. When considered at the individual cell level unit, when luminal glucose concentration is high > 30mM, increased apical membrane SGLT1densities will not greatly alter net transcellular absorption, as this becomes rate limited by GLUT2 densities in the basolateral membranes and GLUT2 reaches near saturation due to the high cytosolic glucose concentration. Additionally, as already explained, increased apical GLUT2 expression reduces net transcellular glucose flux when viewed from the local perspective i.e. cm-2 of intestine. The observed increase in the total area of intestinal SGLT1 distribution following enhanced carbohydrate intake becomes an important factor in total glucose absorption (Ferraris et al. 1992; Moran et al. 2010). The extent of intestinal SGLT1 and GLUT2 distribution increases both along the crypt-villus axis and along the small intestinal longitudinal axis with enrichment of carbohydrate intake. As previously discussed apical GLUT2 expression will spread the luminal glucose load over a wider area, so the increased GLUT2 expression along with SGLT1 could lead to higher rates integrated rate of glucose absorption. This, excessive glucose load, as has been noted already may lead to chronic liver damage. References Ait-Omar, A. et al., 2011. GLUT2 accumulation in enterocyte apical and intracellular membranes: a study in morbidly obese human subjects and ob/ob and high fat-fed mice. Diabetes, 60(10), pp.2598–607. Alexander, B., Cottam, H. & Naftalin, R., 2001. Hepatic arterial perfusion regulates portal venous flow between hepatic sinusoids and intrahepatic shunts in the normal rat liver in vitro. Pflügers Archiv : European journal of physiology, 443(2), pp.257–64. Alexander, B., Rogers, C. & Naftalin, R., 2002. Hepatic arterial perfusion decreases intrahepatic shunting and maintains glucose uptake in the rat liver. Pflügers Archiv : European journal of physiology, 444(1-2), pp.291–8. Caviedes-Vidal, E. et al., 2007. The digestive adaptation of flying vertebrates: high intestinal paracellular absorption compensates for smaller guts. Proceedings of the National Academy of Sciences of the United States of America, 104(48), pp.19132–19137.Ferraris, R.P. et al., 1992. Effect of diet on glucose transporter site density along the intestinal crypt-villus axis. The American journal of physiology, 262, pp.G1060–G1068.Karasov, W.H. & Cork, S.J., 1994. Glucose absorption by a nectarivorous bird: the passive pathway is paramount. The American journal of physiology, 267, pp.G18–G26.Kellett, G.L. & Helliwell, P.A., 2000. glucose-induced recruitment of GLUT2 to the brush-border membrane. Biochem J, 162, pp.155–162.Kimmich, G.A., 1968. Active Sugar Accumulation by Isolated Intestinal Epithelial Cells . Biochemistry, 9, pp.3669–3677.Li, X. et al., 2003. Location and function of intrahepatic shunts in anaesthetised rats. Gut, 52(9), pp.1339–46. McWhorter, T.J. et al., 2013. Paracellular Absorption Is Relatively Low in the Herbivorous Egyptian Spiny-Tailed Lizard, Uromastyx aegyptia. PLoS ONE, 8(4).Moran, A.W. et al., 2010. Expression of Na+/glucose co-transporter 1 (SGLT1) in the intestine of piglets weaned to different concentrations of dietary carbohydrate. The British journal of nutrition, 104(5), pp.647–55. Pappenheimer, J.R., 1998. Scaling of dimensions of small intestines in non-ruminant eutherian mammals and its significance for absorptive mechanisms. Comparative Biochemistry and Physiology - A Molecular and Integrative Physiology, 121, pp.45–58.Soergel, K.H., Whalen, G.E. & Harris, J. a, 1968. Passive movement of water and sodium across the human small intestinal mucosa. Journal of applied physiology (Bethesda, Md. : 1985), 24(1), pp.40–48.Vanis, L. et al., 2011. Effects of small intestinal glucose load on blood pressure, splanchnic blood flow, glycemia, and GLP-1 release in healthy older subjects. American journal of physiology. Regulatory, integrative and comparative physiology, 300(5), pp.R1524–R1531.Van Wijck, K. et al., 2013. Novel multi-sugar assay for site-specific gastrointestinal permeability analysis: A randomized controlled crossover trial. Clinical Nutrition, 32(2), pp.245–251." } ] } ]	1	https://f1000research.com/articles/3-304
https://f1000research.com/articles/3-290/v1	28 Nov 14	{ "type": "Research Article", "title": "Fourier transform infrared spectroscopy study of ligand photodissociation and migration in inducible nitric oxide synthase", "authors": [ "Michael Horn", "Karin Nienhaus", "Gerd Ulrich Nienhaus", "Michael Horn", "Karin Nienhaus" ], "abstract": "Inducible nitric oxide synthase (iNOS) is a homodimeric heme enzyme that catalyzes the formation of nitric oxide (NO) from dioxygen and L-arginine (L-Arg) in a two-step process. The produced NO can either diffuse out of the heme pocket into the surroundings or it can rebind to the heme iron and inhibit enzyme action. Here we have employed Fourier transform infrared (FTIR) photolysis difference spectroscopy at cryogenic temperatures, using the carbon monoxide (CO) and NO stretching bands as local probes of the active site of iNOS. Characteristic changes were observed in the spectra of the heme-bound ligands upon binding of the cofactors. Unlike photolyzed CO, which becomes trapped in well-defined orientations, as indicated by sharp photoproduct bands, photoproduct bands of NO photodissociated from the ferric heme iron were not visible, indicating that NO does not reside in the protein interior in a well-defined location or orientation. This may be favorable for NO release from the enzyme during catalysis because it reduces self-inhibition. Moreover, we used temperature derivative spectroscopy (TDS) with FTIR monitoring to explore the dynamics of NO and carbon monoxide (CO) inside iNOS after photodissociation at cryogenic temperatures. Only a single kinetic photoproduct state was revealed, but no secondary docking sites as in hemoglobins. Interestingly, we observed that intense illumination of six-coordinate ferrous iNOSoxy-NO ruptures the bond between the heme iron and the proximal thiolate to yield five-coordinate ferric iNOSoxy-NO, demonstrating the strong trans effect of the heme-bound NO.", "keywords": [ "iNOS", "migration", "ligand", "heme binding", "FTIR" ], "content": "Introduction\n\nNitric oxide synthases (NOSs) are homodimeric heme enzymes that catalyze the oxidative degradation of L-arginine (L-Arg) to nitric oxide (NO)1,2. Three structurally similar NOS isoforms have been identified in endothelial cells (eNOS), neuronal tissues (nNOS) and in macrophages (iNOS)3. Different from eNOS and nNOS, iNOS is not expressed in resting cells but induced upon inflammatory and immunologic stimulation. Each NOS protomer consists of an oxygenase and a reductase domain. In the catalytic oxygenase domain (NOSoxy), dioxygen (O2) binds to a central heme prosthetic group, anchored to the polypeptide chain via a proximal cysteine residue (Figure 1). Its thiol sulfur atom accepts a hydrogen bond from an adjacent tryptophan residue. The substrate, L-Arg, is accommodated directly on top of the heme plane in the distal pocket; the cofactor tetrahydrobiopterin, H4B, binds along the side of the heme4–7. L-Arg and H4B are linked through an extended hydrogen bonding network mediated by one of the heme propionate groups. The reductase domain, NOSred, binds flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and reduced nicotinamide adenine dinucleotide phosphate (NADPH). It provides the electrons for the catalytic reaction proceeding in the oxygenase domain. In a first step, L-Arg is converted to N-hydroxy L-Arg (NOHA). Subsequently, NOHA is decomposed into citrulline and nitric oxide (NO). Electron transfer is enabled by calmodulin binding in the interface between the two domains8.\n\nThe CO ligand was added for illustration.\n\nThe NO molecule generated during enzymatic turnover can either coordinate directly to the heme iron or diffuse out of the protein into the environment. From there, it may again bind in a bimolecular process9. Formation of the very stable ferrous NO complex results in self-inhibition of the enzyme. The probability of forming this product depends on the dissociation rate coefficient of NO from the ferric heme, the likelihood of autoreduction of the ferric NO-bound form to the ferrous derivative with its much stronger NO affinity, and the probability of oxidizing the ferrous NO-bound species to the ferric form plus nitrate by O210. Deactivation of the enzyme may also occur via nitrosylation of the side chains of two cysteine residues coordinating a zinc ion in the dimer interface, which leads to irreversible dissociation into non-functional monomers11–14.\n\nThe iNOS isoform has been implicated in the pathogenesis of various diseases; so there is a growing interest in developing potent and highly selective inhibitors15,16. Their targeted design requires detailed insights into the interactions between ligand, substrate and the surrounding protein matrix. Therefore, we have investigated ligand and substrate binding in the iNOS oxygenase domain, iNOSoxy, by using Fourier transform infrared (FTIR) spectroscopy of the stretching vibrations of carbon monoxide (CO) and NO as ligands rather than the physiological ligand O2. They are of similar size as O2, which suggests that ligand dynamics within the protein may be comparable for all three ligands. CO and NO both have excellent properties as infrared (IR) spectroscopic probes17. CO has proven to be an attractive heme ligand because the CO bond stretching vibration gives rise to strong mid-IR absorption bands that can be measured with exquisite sensitivity and precision17,18. The IR bands are fine-tuned by electrostatic interactions with the environment19–21; therefore, CO is frequently utilized as a local probe of protein structure and dynamics22.\n\nIn the gas phase, CO absorbs at 2143 cm-123. When bound to the central iron of a heme cofactor, the CO stretching frequency, νCO, which is typically in the 1900 – 2000 cm-1 spectral range, is susceptible to changes in the iron-ligand bond and the local electric field due to the vibrational Stark effect24–29. There are two major contributions to the heme iron-CO bond, i.e., σ-donation from a weakly antibonding 5σ MO of CO to the iron 4s and 3dz2 orbitals and π-backbonding from the iron 3dz orbitals to the strongly antibonding CO 2π* orbital30. A positive charge located near the CO oxygen attracts electron density, causing a decrease in σ-donation and an increase in backbonding. Consequently, the C–O bond order is reduced and νCO shifts to lower values19–21. A negative charge has the opposite effect.\n\nAfter photodissociation of CO-bound heme protein samples, the stretching bands of unbound CO trapped inside a protein are found within the range from ~2080 to ~2160 cm-118,31. The vibrational bands can reveal changes related to ligand relocation to other sites within the protein18,29,32,33, rotational motions of the ligand in these sites25,34 and protein conformational changes35. Often, there are doublets of bands corresponding to opposite orientations of the CO at a particular transient docking site27,29,32,36–38. The bond order and, therefore, νCO increases if the carbon atom interacts with a hydrogen bond donor, whereas an interaction with the ligand oxygen reduces both the bond strength and the stretching frequency29.\n\nUnlike CO, which only binds to a ferrous (FeII) heme iron, NO may coordinate to both the ferrous and the ferric (FeIII) forms. So far, FTIR studies using NO have remained scarce because of its weaker intrinsic absorption. Furthermore, there is spectral overlap with the amide bands and ultrafast recombination of a major fraction of proteins even at very low temperatures. Therefore, only small photoproduct yields are obtained in experiments probing longer times such as FTIR, which renders experiments with ferrous NO technically challenging. Consequently, we have limited ourselves to NO binding to ferric heme in this work. For iNOS, this complex is of physiological relevance because the heme iron is in the ferric state after completion of the catalytic cycle.\n\nHere, we have performed FTIR studies on iNOS at cryogenic temperatures, at which ligand rebinding is very slow. Thus, photoproducts induced by illumination are long-lived and can be conveniently studied by photolysis difference spectroscopy. Moreover, essentially all protein (and solvent) motions are frozen in39,40, so the ligands cannot escape to the solvent and can be observed within the protein matrix. We have combined FTIR with temperature-derivative spectroscopy (TDS)41–43, which allows us to disentangle photolysis-induced absorption changes caused by the different types of ligand dynamics.\n\n\nMaterials and methods\n\nThe iNOSoxy domain, with its first 65 residues deleted (Δ65 iNOSoxy, referred to as iNOSoxy in the following), was expressed essentially as described44. Briefly, iNOSoxy containing plasmids (pCWori) were transformed into competent Escherichia coli cells (strain BL21). The cells were plated on agar in the presence of 390 µM ampicillin (Carl Roth, Karlsruhe, Germany) and cultured overnight at 37°C. A single colony was added to 150 ml terrific broth (TB, Carl Roth) supplemented with ampicillin (390 µM) and agitated for 12 h at 37°C and 250 rpm. 10 ml of the overnight culture were added to 1.5 l TB, containing 390 µM ampicillin, and grown to an optical density of ~1 at 600 nm. Then, the temperature was lowered to 30°C and δ-aminolevulinic acid (44 µM, Sigma-Aldrich, St. Louis, MO, USA) and hemin (8 µM, Sigma-Aldrich) were added. iNOS expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG, Carl Roth) to a final concentration of 100 µM. After 48 h (fresh ampicillin was added every 16 h), the cells were harvested by centrifugation for 20 min at 4°C and 2,000 rpm (swing-bucket rotor, 4–16 K, Sigma, Osterode, Germany). The cells were resuspended in lysis buffer (40 mM HEPES, 10% glycerol (vol.), 200 mM NaCl, pH 7.6, Carl Roth), mixed with 2 mg DNase (Sigma-Aldrich), and ruptured using a bead-beater (Biospec, Bartlesville, USA), filled with 0.1 mm (diameter) zirconia/silica beads (three treatments of 2 min each). The lysate was separated from the beads by a glass filter and loaded onto an immobilized-metal ion affinity column equilibrated with lysis buffer (Ni Sepharose 6 FastFlow, GE Healthcare). After washing with lysis buffer supplemented with increasing concentrations of imidazole (0, 10, 40 mM, Sigma-Aldrich), the protein was eluted with lysis buffer containing 160 mM imidazole. Appropriate fractions were pooled, dialyzed against water and concentrated by using Vivaspin Turbo 15 (cut-off 10 kDa) centrifugal concentrators (Sartorius, Göttingen, Germany). Finally, the protein was lyophilized and stored at -20°C.\n\nTo prepare CO-ligated iNOSoxy, 12 mg freeze-dried iNOS were slowly added to 40 µl cryosolvent (75%/25% glycerol/100 mM potassium phosphate buffer (v/v), pH 7.4, and, if so desired, supplemented with L-Arg and NOHA substrate (Sigma-Aldrich) or H4B cofactor (Sigma-Aldrich) to reach final concentrations of 200 mM and 100 mM, respectively) and stirred under 1 atm CO for 60 min. Subsequently, a two-fold molar excess of an anaerobically prepared sodium dithionite solution (Sigma-Aldrich) was added with a gas-tight Hamilton syringe, and the solution was stirred for another 10 min. To remove any undissolved protein, the solution was centrifuged for 10 min at 13,400 rpm (Minispin centrifuge, Eppendorf, Hamburg, Germany) before loading it into the sample cell. For an NO-ligated sample, ferric iNOSoxy was dissolved in cryosolvent and stirred under an N2 atmosphere for 1 h. The gas phase above the sample was replaced repeatedly by N2 to efficiently remove O2. Finally, a few microliters of NO gas were added with a gas-tight syringe. NO ligation to the heme iron was confirmed by UV/vis absorption spectroscopy.\n\nA few microliters of the sample solution were sandwiched between two CaF2 windows (diameter 25.4 mm) separated by a Mylar washer. The windows were mounted inside a block of oxygen-free high-conductivity copper. The copper block was attached to the cold-finger of a closed-cycle helium refrigerator (model F-50, Sumitomo, Tokyo, Japan). The sample temperature was measured with a silicon temperature sensor diode and regulated in the range 3 – 320 K by a digital temperature controller (model 336, Lake Shore Cryotronics, Westerville, OH). A continuous-wave, frequency-doubled Nd-YAG laser (Samba, Cobolt, Solna, Sweden), emitting up to 300 mW output power at 532 nm, was used to photolyze the sample. The laser beam was split and focused with lenses on the sample from both sides. Transmission spectra were recorded on a Vertex 80v FTIR spectrometer (Bruker, Karlsruhe, Germany) at a resolution of 2 cm–1, using either an InSb detector (75 µm thick Mylar, 1,700 to 2,300 cm–1) or an MCT detector (<5 µm thick Mylar, 1,100 to 2,300 cm–1).\n\nThe infrared absorption of CO and NO can be studied selectively by using photolysis difference spectroscopy, which involves measurement of IR transmission spectra, I(ν, T), before and after photolysis. The difference absorbance of the two spectra, ΔA(ν, T) = log(Idark/Ilight), contains only features that are due to photodissociation of the ligand from the heme iron. The missing absorption of the heme-bound ligands (A bands) after photolysis and the corresponding absorption of the photolyzed ligands (photoproduct bands) are displayed with negative and positive amplitudes, respectively. Peak positions and fractional occupancies were determined by fits with Gaussian band shapes; they are compiled in Table 1. In the following, we use the Gaussian band positions (frequencies) at 4 K as a subscript to ‘A’ (denoting the heme-bound state) to distinguish the absorbance bands and also to refer to a particular substate of the protein.\n\nDifferent illumination protocols were applied for photodissociation17. Before starting a TDS experiment, the sample was illuminated for 10 s at 4 K to trap the photolyzed ligand close to the heme iron at the so-called primary docking site B. Alternatively, under ‘slow-cool’ illumination, the sample was cooled from 160 to 4 K at a rate 0.3 K/min under constant laser illumination to enable the photodissociated ligands to sample alternative docking sites that may not be accessible upon photolysis at 4 K. In both protocols, 300 mW laser power at 532 nm was used. To monitor the photodissociation kinetics, the samples were continuously illuminated for 15,000 s at reduced laser power (0.3 mW or 10 mW), and transmission spectra were recorded continuously. For comparison, the photolysis yield was scaled with respect to complete photodissociation with full laser power (300 mW).\n\nTDS, an experimental protocol designed to study thermally activated rate processes involving enthalpy barrier distributions, has been described in great detail elsewhere41–43. Briefly, a non-equilibrium state is created in the sample at low temperature, e.g., by photolysis with visible light. The integrated absorbance, A, of a spectral band taken at the lowest temperature represents the total photolyzed population, N. Subsequently, thermal relaxation of the sample back to equilibrium is recorded while the sample temperature is ramped up linearly over a few hours in the dark. One FTIR transmission spectrum is taken for every 1-K temperature increase. In the simplest analysis, we assume that any change in integrated absorbance is due to ligand rebinding and, therefore, proportional to a population change, ΔN, during acquisition of two successive spectra. TDS data are conveniently presented as two-dimensional contour plots, with solid lines indicating an absorbance increase and dashed lines a decrease. Contours are spaced logarithmically to emphasize small features.\n\n\nResults and discussion\n\nIn the following, we present 4-K FTIR photolysis difference spectra of iNOSoxy-CO and briefly discuss the influence of substrate, substrate intermediate and cofactor on the CO stretching vibration and rebinding. For additional information, we refer to Jung et al.45 and Li et al.46.\n\nPhotolysis difference spectra at 4 K. The 4-K absorption difference spectrum of iNOSoxy-CO displays two broad, extensively overlapping A bands at 1945 and 1959 cm–1, indicative of two active site subconformations with significant intrinsic structural heterogeneity (Figure 2a). Adding the H4B cofactor induces only small changes; the resulting spectrum can be described by a dominant A band centered on 1951 cm-1 and a minor one at 1924 cm-1 (Figure 2a). As H4B binds along the side of the heme4 and, thus, not in the immediate vicinity of the heme-bound CO, it is not expected to modify νCO to any significant extent. In contrast, the presence of L-Arg shifts the main A band of iNOSoxy-CO/L-Arg to 1904 cm-1; smaller features are located at 1921 and 1951 cm-1 (Figure 2a). The pronounced red-shift of A1904 arises from the electron-withdrawing effect of the terminal, positively charged NH2+ moiety of the L-Arg side chain close to the bound CO4. The position of the A1921 band is indicative of an electrostatic interaction of the CO dipole with a less pronounced positive partial charge, most likely the neutral terminal amino group of the L-Arg side chain.\n\nIf the reaction intermediate NOHA is present, three A bands at 1903, 1937 and 1956 cm-1 are discernable (Figure 2a). The crystal structure shows that NOHA binds in the same orientation in the active site as L-Arg, with the side chain pointing towards the heme iron47. Therefore, we suggest that, in those iNOSoxy molecules absorbing within the A1937 band, a hydrogen bonding interaction exists between the CO ligand and the hydroxyl group of the NOHA side chain. A1903 is most likely associated with iNOSoxy molecules, in which the terminal amine of the NOHA side chain is protonated (pK = 8.148) and points towards the heme-bound CO. The protonated NOHA has been suggested to be the catalytically active substrate intermediate49,50.\n\nThe absorption spectra of photolyzed CO are plotted in Figures 2b (brief illumination at 4 K) and 2c (slow-cool illumination); peak positions and relative areas are included in Table 1. For comparison, the integrated absorption in each spectral region was scaled to the same area. We note that the ratio of the integrated areas of the A and photoproduct bands is ~2018.\n\nAll photoproduct spectra obtained after 10-s illumination at 4 K have absorption bands in the 2120 – 2130 cm-1 spectral range (Figure 2b). The spectrum of iNOSoxy-CO is composed of two stretching bands at 2124 and 2129 cm-1. With H4B, photoproduct bands appear at 2124 and 2133 cm-1, indicating that the cofactor has an effect on νCO of the unbound CO. In the presence of L-Arg, the absorption bands are centered on 2120 and 2131 cm-1, and there are two additional bands at 2144 and 2150 cm-1. Their higher stretching frequencies suggest formation of a hydrogen bond between the ligand carbon and the terminal amine group of L-Arg29. Upon NOHA binding, the photoproduct bands are centered on 2122 and 2133 cm-1. The minor absorption at 2145 cm-1 can be associated with CO ligands photolyzed from iNOSoxy/NOHA trapped in its A1903 conformation.\n\nThe photoproduct spectra obtained after slow-cool illumination (Figure 2c) are similar to the ones recorded after 10-s illumination (Figure 2b), suggesting that it is not possible to populate additional docking sites to any significant extent. The greatest difference is seen for iNOSoxy-CO. Its photoproduct spectrum shows two well separated bands at 2124 and 2134 cm-1 rather than the non-separated doublet seen in Figure 2b. We also note that there is an additional shoulder at 2117 cm-1 for iNOSoxy-CO/NOHA.\n\n(a) Absorption of the heme-bound CO. (b) Photoproduct bands obtained after 10-s illumination at 4 K. (c) Photoproduct bands obtained after constant illumination during slow cooling from 160 to 4 K.\n\nCO rebinding in iNOSoxy. To obtain more information on the photoproduct states, TDS measurements were started at 4 K immediately after illumination. Figure 3 displays the contour maps obtained after 10-s illumination at 4 K, with the absorption changes in the A bands and the photoproduct bands in the left and right columns, respectively.\n\nLeft column: Absorption changes in the bands of heme-bound CO. Right column: Absorption changes in the photoproduct bands. Contours are spaced logarithmically; solid and dotted lines represent increasing and decreasing absorption, respectively. iNOSoxy-CO (a, b) w/o substrate; (c, d) with H4B; (d, e) with L-Arg; (f, g) with NOHA.\n\nAll iNOSoxy-CO samples display single-step CO rebinding. This observation indicates that there is only a single kinetic state of the photolyzed protein-ligand complex, and the presence of sharp photoproduct bands indicates that the photolyzed ligands are trapped in transient docking sites with well-defined orientations. In substrate-free iNOSoxy-CO, recombination is already maximal at 4 K and extends to ~70 K (Figure 3a, b). Rebinding in the dominant A1951 substate of iNOSoxy-CO/H4B peaks at 20 K; as in iNOSoxy-CO, the process extends to 70 K. Only the minor A1924 subpopulation shows a focused rebinding peak at ~60 K (Figure 3c). The photoproduct map does not yield additional information (Figure 3d). Binding of either L-Arg or NOHA in the active site shifts CO rebinding to higher temperatures, suggesting that the hydrogen bonding interaction stabilizes the ligands at the transient docking site against rebinding (Figures 3e and 3g). Maximal rebinding in iNOSoxy/NOHA, i.e., in A1903 and A1937, occurs at 50 – 60 K (Figure 3g). The corresponding photoproduct bands are centered on 2122 and 2133 cm-1 (Figure 3h). The contours at 1950 – 1960 cm-1 (Figure 3g) represent rebinding in the NOHA-free A1956 substate. With L-Arg anchored in the active site, CO ligands return to the heme iron also at ~50 – 60 K (Figure 3e). The corresponding photoproduct map shows a concomitant loss of the photoproduct bands at 2150, 2144, 2131 and 2120 cm-1, associating these bands with CO molecules trapped in the vicinity of the substrate (Figure 3f). A population transfer between photoproduct states due to CO rotation32,51,52 is apparent from the mirror-imaged dashed and solid contours at 2131 and 2144 cm-1 at 12 K.\n\nThe TDS maps after slow-cool illumination (Figure 4) show only marginal differences to the ones obtained after brief 4-K illumination (Figure 3), which confirms that the photodissociated CO ligands populate only a single kinetic state. Notably, after slow-cool illumination, rebinding generally occurs at slightly higher temperatures than after brief 4-K illumination. The observed slowing may be attributed to small structural changes near the active site, causing an increase of the ligand binding barrier. A similar effect was also visible in MbCO upon extended illumination below 40 K42 as well as in NO- and CO-ligated nitrophorin 435.\n\nLeft column: Absorption changes in the bands of heme-bound CO. Right column: Absorption changes in the photoproduct bands. Contours are spaced logarithmically; solid and dotted lines represent increasing and decreasing absorption, respectively. iNOSoxy-CO (a, b) w/o substrate; (c, d) with H4B; (d, e) with L-Arg; (f, g) with NOHA.\n\nIn a typical globin protein involved in ligand transport or storage, the primary ligand docking site B is indispensable because it ensures efficient ligand binding to and release from the heme iron53. Incoming ligands are ‘caught’ in site B before the actual bond formation process occurs32,54. Upon thermal dissociation from the heme iron, ligands can remain unbound in site B for some time, which increases their probability to escape from the protein. Without this site, they would immediately recombine with the heme iron, as is, e.g., observed for NO-transporting nitrophorin35 and modified cytochrome c55.\n\nThe catalytic reaction of iNOS requires sequential binding of two O2 molecules and efficient release of the NO product. Therefore, the B site is likely to have dual functionality. On the one hand, it allows efficient O2 binding to the heme iron. On the other hand, it ensures efficient release of the generated NO. Using CO as a ligand, we have shown that the B site is readily accessible for ligands photodissociated from the heme iron, both in the presence and absence of L-Arg or NOHA. The substrates stabilize the CO ligand at the transient site via hydrogen bonding. This stabilizing effect is also seen for the minor A1924 subpopulation of iNOSoxy/H4B. Presumably, a small fraction of H4B molecules are positioned such that they can form a direct hydrogen bond.\n\nThe NO stretching absorption is also very suitable as a local probe of the active site structure and of ligand movements within a protein17. Despite their similar sizes, the ligands may show different dynamics inside the protein56. For example, in myoglobin (Mb), a transient docking site on the proximal side of the heme is readily populated by CO but not at all by NO56. Such subtle differences could be relevant for the inhibitory effects of NO. Therefore, we have analyzed NO binding in ferric iNOSoxy using FTIR-TDS at cryogenic temperatures.\n\nPhotolysis difference spectra at 4 K. Figure 5 displays 4-K photolysis difference spectra of various ferric iNOSoxy-NO preparations. Most spectra show an A band at 1870 cm-1 associated with NO bound in an active site without bound cofactor or substrate (Table 1). In the spectrum of iNOSoxy-NO, A1870 is rather broad, suggesting significant conformational heterogeneity at the active site. The spectrum of iNOSoxy-NO/NOHA is very similar, dominated by the broad A1870 band; the only clear change from iNOSoxy-NO is a shoulder at 1851 cm-1. This comparison suggests that NOHA is bound only in a small subfraction reflected by the shoulder. In iNOSoxy-NO/L-Arg, A1847 and A1829 report the binding of L-Arg. A1870 is still present due to incomplete saturation with substrate (Figure 5). Interestingly, Rousseau et al.2 could not identify any changes of νFe-N in their resonance Raman spectra upon binding of L-Arg and even hypothesized that L-Arg does not bind to ferric iNOSoxy-NO. With H4B anchored next to the heme, the A band is shifted to 1872 cm-1, and another absorption band emerges at 1890 cm-1.\n\nThe stretching bands of heme-bound NO (NO after laser illumination at 300 mW) are plotted with negative (positive) amplitude. A bands of the NOHA spectrum were scaled independently of photoproducts (factor 2.07) to match the A bands of the spectrum without substrate. Dotted line: 4-K photolysis difference spectrum of iNOSoxy-NO/L-Arg, obtained upon illumination at 0.3 mW. Dashed line: iNOSoxy-NO/L-Arg photoproduct spectrum (obtained by calculating the difference between the two iNOSoxy-NO/L-Arg spectra). Inset: extended 4-K photolysis difference spectrum of iNOSoxy-NO.\n\nMost of the observed spectral shifts can again be explained by backbonding57 because the ferric NO-ligated ground state, which is best described as FeIINO+, is isoelectronic to FeIICO58. The heme-bound NO absorbs at 1870 cm-1. L-Arg shifts νNO to lower frequencies; the A1829 and A1847 bands indicate an interaction between the NO and the positively charged and neutral terminal amino groups of the L-Arg side chain. As already observed for CO, the effect of NOHA is less pronounced; its presence is visible via a shift of the A band to 1851 cm-1. Interestingly, the NO stretching absorption is also affected by H4B. The band shifts slightly and, in addition, it becomes rather narrow, which is indicative of a more homogeneous active site environment or restricted dynamics of the heme-bound NO due to the bound H4B35,59. In 2005, Rousseau et al.2 reported that, upon H4B binding, a Raman band emerges that was assigned to the Fe-N-O bending mode, δFe-N-O, of the ferric adduct, indicating a more homogeneous bending of the bent NO. In thiolate-ligated FeIIINO adducts, NO is typically bound at an angle of 160°60–66, and H4B binding next to the heme is not expected to modify this angle due to steric interactions. It may, however, restrict its librational dynamics around this angle, possibly because of the increased heme distortion caused by H4B67,68. The additional band at 1890 cm-1 may indicate partial occupancy of a water molecule in the active site62.\n\nThe photoproduct bands, displayed in Figure 5 with positive amplitudes, are in the 1810 – 1830 cm-1 spectral range and, thus, red-shifted by only ~50 cm-1 from those of the heme-bound NO (Table 1). For iNOSoxy-NO/L-Arg, the photoproduct and A bands even overlap. Their decomposition (details are discussed below) yields a narrow photoproduct band at 1814 cm-1 and a broad feature at 1822 cm-1. iNOSoxy-NO and iNOSoxy-NO/NOHA show two photoproduct bands at 1814 and 1818 cm-1. Interestingly, these bands are about as strong as the A bands, which strongly suggests that they do not represent unbound NO trapped in a transient docking site but rather heme-bound NO with restricted librational freedom.\n\nIn contrast to all other samples, the iNOSoxy-NO/H4B photoproduct spectrum reveals only a very weak feature at ~1818 cm-1. This finding may be explained by a photolyzed NO that cannot be trapped in well-defined orientations. As a result, the stretching absorption becomes extremely broad and hardly distinguishable from the background. A similar effect was observed for NO in the primary photoproduct site B of ferric Mb56.\n\nNO rebinding in ferric iNOSoxy. To gain additional information on the peculiar, strongly absorbing NO photoproduct bands, TDS experiments were started immediately after illuminating NO-ligated samples at 4 K. Figure 6 displays the absorption changes in the A bands and in the photoproduct bands with solid and dotted lines, respectively. The contour maps obtained after slow cool illumination (not shown) are essentially identical, as for the CO-ligated samples.\n\niNOSoxy-NO (a) w/o substrate; (b) with H4B; (c) with L-Arg; (d) with NOHA. Contours are spaced logarithmically; solid and dotted lines represent increasing and decreasing absorption, respectively.\n\nIn iNOSoxy-NO, NO rebinding in A1870 starts already at the lowest temperatures (Figure 6a) and extends to ~90 K. The decay of the photoproduct, however, occurs predominantly between 80 and 120 K, indicating that these bands cannot be associated with NO ligands photolyzed from the ferric heme iron, as reported by the A1870 band. Apparently, laser illumination produces a photoproduct band from another NO species in the sample. The TDS map of iNOSoxy-NO/NOHA (Figure 6d) shows essentially the same features. It is likewise evident that NO rebinding is complete below 80 K, whereas the strange photoproduct feature disappears in the temperature range 80 – 120 K. In iNOSoxy-NO/H4B (Figure 6b), NO rebinding at the ferric iron also starts at 4 K. In a subpopulation, recombination peaks at ~65 K; absorption changes of photoproducts are too small to be detected. NO rebinding in the L-Arg-bound A1829 and A1847 substates occurs mainly below 30 K, concomitantly with the decay of the photoproduct (Figure 6c). The apparent maximum in the contours at 15 K and ~1850 cm-1 is artificial and results from the superposition of the A bands and the photoproduct bands (compare Figure 5). Recombination in the substrate-free A1870 fraction of the sample is maximal at 4 K and extends out to ~70 K, consistent with the data shown in Figure 6a.\n\nIn summary, rebinding of NO to the ferric heme of iNOSoxy is a one-step process. The corresponding photoproduct bands, i.e., the absorption bands of NO photodissociated from the ferric heme, were not identifiable. Presumably, NO is bound only weakly within the protein, without any well-defined orientation and without any additional stabilization via hydrogen bonding interactions to the substrate or the cofactor. As a consequence, the NO has a broad stretching absorption that cannot be distinguished from the background. Note that, if the photoproduct bands were masked by the strong bands at ~1820 cm-1, they should have become visible in the spectrum of iNOSoxy-NO/H4B (Figure 5).\n\nIdentification of the iNOSoxy-NO photoproduct. The TDS data in Figure 5 clearly prove that the strong absorption bands at ~1820 cm-1 are not generated by photodissociation of NO bound to ferric heme, absorbing at ~1870 cm-1. To identify the corresponding pre-illumination states, we screened the 4-K FTIR photolysis difference spectrum of iNOSoxy-NO from 1,100 to 2,300 cm-1 and detected a band at 1616 cm-1, which we tentatively associate with a six-coordinate (6C) ferrous NO adduct (Figure 5, inset). This assignment is supported by the νNO of 1591 cm-1 reported for 6C ferrous P450cam-NO69. Praneeth et al.70 also computed frequencies in this range, νNO = 1617 cm-1 and νNO < 1600 cm-1 for thiophenolate- and alkylthiolate-heme complexes, respectively, using density functional theory calculations on ferrous, thiolate-coordinated porphyrin model systems.\n\nThe admixture of a ferrous NO species in our samples does not come as a surprise. Ferric iNOSoxy-NO is unstable and spontaneously converts to a ferrous 6C NO-ligated species. This conversion may take place during loading and cooling of an FTIR sample, which typically takes ~2 h. This species may subsequently evolve further to a five-coordinate (5C) complex by dissociation of the thiolate ligand on time scales of minutes to hours, depending on the iNOSoxy oligomerization state67,71–73. Here, we can safely exclude formation of significant amounts of 5C ferrous iNOSoxy-NO because we have not observed the characteristic IR bands of this species at ~1670 cm-153.\n\nNO photodissociation from the 6C adduct is not expected to generate NO photoproduct bands that are of similar strength as the original A1616 band. Therefore, there must be yet another species responsible for the strong absorption at ~1820 cm-1. Perhaps, light-induced breakage of the iron-sulfur rather than the iron-NO bond could lead to an alternative photoproduct, considering the strong trans effect exerted by the NO in 6C ferrous heme NO adducts66. Indeed, Ibrahim et al.74 had noticed earlier that laser light passing through solution samples of 6C ferrous model porphyrins adducts during resonance Raman measurements was sufficient to photodissociate the axial thiolate base trans to the NO75. This effect could be suppressed by lowering the temperature to 77 K and reducing the laser power. Accordingly, we have illuminated the iNOSoxy-NO/L-Arg sample at low laser intensity (0.3 mW at 532 nm). This power was still sufficient to photodissociate the NO from the 6C ferric heme adduct (dotted line in Figure 5), photoproduct bands at ~1820 cm-1, however, did not emerge, confirming that the photoproduct was not formed. Therefore, we propose that illumination of 6C ferrous iNOSoxy-NO with sufficient laser power leads to rupture of the bond between the iron and the proximal Cys194 thiolate, leaving behind a 5C iNOSoxy-NO. Because the NO is still bound to the heme iron, the intensity of the IR bands at ~1820 cm-1 is comparable to that of other A bands25,34. The NO stretching frequency of the 5C adduct indicates that the ligand is coordinated to a ferric iron, so that the Cys194 sulfur is negatively charged after photodissociation. Similar NO stretching frequencies were reported for an isolated 5C ferric heme nitrosyl complex (νNO = 1842 cm-176) and for NO-ligated porphyrins with phenyl (νNO = 1825 cm-1) and pentafluorophenyl (νNO = 1859 cm-1) substituents on the four meso positions77. If the laser power is sufficiently high (300 mW), it is even possible to photodissociate the NO from the 5C ferric iNOSoxy-NO, leaving behind a four-coordinate, ‘naked’ heme as a ‘secondary photoproduct’ (Figure 7a).\n\nTemporal development of the integrated absorbance of the bands of 5C ferric iNOSoxy-NO (open symbols) and 6C ferric iNOSoxy-NO (filled symbols) (a, c) during constant illumination at 4 K (circles: 10 mW, 532 nm; triangles: 300 mW, 532 nm) and (b, d) after the laser was switched off. Black: iNOSoxy-NO; red: iNOSoxy-NO/L-Arg.\n\nL-Arg binding in the active site lowers the yield of ferric 5C iNOSoxy-NO upon laser illumination (Figures 7a and c) and favors reformation of the iron-sulfur bond as soon as the laser is switched off (Figures 7b and d). This effect may result from the competition between the NO ligand and the thiolate for σ charge donation to the heme iron; the higher the donation, the stronger the bond to the donor and the weaker the bond to the opposing heme ligand. The σ donor strength of the thiolate is altered by hydrogen bonding interactions to the sulfur atom66. Using sulfur K-edge x-ray absorption spectroscopy and density functional theory calculations, Dey et al.78 showed that each hydrogen bond reduces the electron-donating power of the thiolate sulfur. The NO electron donor ability and, therefore, its repulsive trans effect can be reduced by interactions that draw electron density away from the NO79,80, here by the hydrogen bonding interaction with L-Arg, so that the axial iron-sulfur bond is stabilized.\n\nWe also note that 6C ferrous iNOSoxy-NO is not stable in the presence of H4B but spontaneously oxidizes to the ferric form46. Consequently, the yield of the 5C adduct is negligible, as is indicated by the low intensity of the absorption bands (Figure 5).\n\nFerric 5C iNOSoxy-NO. In view of the competition between the NO ligand and the thiolate for σ charge donation to the heme iron, one should expect νNO of the 5C photoproduct lacking the thiolate ligand to be blue-shifted with respect to νNO of the 6C adduct because the repulsive trans effect of the thiolate has been removed. Experimentally, however, the opposite behavior is observed (Figure 5). To resolve this apparent discrepancy, one has to consider that the 5C ferric form originates from a 6C ferrous species, in which the NO is typically bound at an angle of ~140°. In the corresponding 6C ferric derivatives, the Fe – N – O angle is normally ~160°. At cryogenic temperatures, the dynamics of the protein matrix is completely arrested39,40.\n\nConsequently, the NO is held in the strongly bent (lower angle) orientation of the 6C ferrous form. Based on DFT calculations, Linder et al.81 reported that reducing the angle from 160° to 150° shifts νNO in 5C model porphyrins from 1897 to 1857 cm-1. Therefore, we suggest that the low νNO of the 5C form is caused by NO binding at a small angle. We note that the similar νNO in 5C and 6C ferric iNOSoxy-NO/L-Arg implies that the bound substrate controls the angle at which the NO binds. Apparently, steric constraints override the bending induced by the trans effects.\n\nFinally, we point out that, in contrast to the photo-induced 6C ferric → 5C ferric transition observed in the FTIR experiments at cryogenic temperatures, the spontaneous conversion of the 6C ferric NO-bound iNOSoxy derivative at physiological temperatures involves two NO molecules and yields a 5C ferrous species71,72,82. After binding the first NO, the ferric 6C iNOSoxy-NO reacts with a second ligand to yield 6C ferrous iNOSoxy-NO. This complex immediately converts to the 5C form and a nitrosonium ion (NO+). The ion may diffuse towards the zinc binding site and nitrosylate one of the Cys residues involved in coordinating the zinc.\n\n\nConclusions\n\nFTIR spectroscopy at cryogenic temperatures, especially in combination with sophisticated illumination and data acquisition temperature protocols, provides quantitative data on protein-ligand interactions. Our FTIR-TDS studies on iNOSoxy have shown that CO and NO rebinding involve only a single transient state in iNOSoxy. The CO is stabilized in well-defined orientations at the docking site by hydrogen bonding interactions and, therefore, gives rise to rather narrow photoproduct bands. In contrast, photoproduct bands associated with the photolyzed NO cannot be resolved. The NO appears to be trapped in less specific orientations, which may favor the release of this ligand. Under physiological conditions, release of the generated NO from the protein is facilitated.\n\nUpon illumination of 6C ferrous iNOSoxy-NO at cryogenic temperatures, a 5C ferric NO adduct was identified, providing direct evidence for light-induced breakage of the iron-thiolate bond. Future studies along these lines are likely to contribute to a better understanding of functional processes in which the NO ligand is involved.\n\n\nData availability\n\nF1000Research: Dataset 1. Fourier transform infrared photolysis difference spectra of CO- and NO-ligated inducible nitric oxide synthase, 10.5256/f1000research.5836.d3948183", "appendix": "Author contributions\n\n\n\nMichael Horn performed the experiments. All authors have contributed to planning the experiments and analyzing the results. All authors were involved in writing and editing the draft of this manuscript. All authors have read and approved the final version.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the Deutsche Forschungsgemeinschaft (Grant Ni291/10). We acknowledge support by Deutsche Forschungsgemeinschaft and Open Access Publishing Fund of Karlsruhe Institute of Technology.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nLi HY, Poulos TL: Structure-function studies on nitric oxide synthases. 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Data Source" }	[ { "id": "6853", "date": "05 Dec 2014", "name": "Pál Ormos", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is investigated how ligands NO and CO dissociate and bind to inducible nitric oxide synthase. The applied method is FTIR difference spectroscopy, in particular low temperature temperature derivative spectroscopy, most appropriate to elucidate details of the process. The binding route has been clarified and compared to related ligand binding heme proteins. The results are important in characterizing the enzyme. As I understand, the task of the referee is primarily to judge the soundness, the technical quality of the work. There is absolutely no problem in this respect: the method is appropriate, executed perfectly, the conclusions are well supported. I suggest indexing without any modification.", "responses": [] }, { "id": "6849", "date": "05 Dec 2014", "name": "Marius Schmidt", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very interesting investigation on protein ligand interaction in iNOS. It is well written and informative. It should be indexed, and requires only minor revisions.p.6 text: ”In substrate free iNOSoxy-CO, recombination is already maximal at 4 K and extends to ~70 K.”What does this mean? What is a maximal recombination?Suggestion: “there is already substantial recombination at 4 K and the process extends to 70 K.” p. 6 text: “A population transfer between photoproduct states due to CO rotation 32,51,52 is apparent from the mirror-imaged dashed and solid contours at 2131 and 2144 cm-1 at 12 K”.Just write a sentence or two why that is so. The non-expert reader should not read the literature for this. Figure 3, caption: there is a mix-up with panel numbers. The case for a 5C and the 4C (naked) heme is well made. This is a very interesting result and merits closer investigation by X-ray structure determination.", "responses": [] } ]	1	https://f1000research.com/articles/3-290
https://f1000research.com/articles/3-253/v1	27 Oct 14	{ "type": "Review", "title": "Rice (Oryza) hemoglobins", "authors": [ "Raúl Arredondo-Peter", "Jose F. Moran", "Gautam Sarath", "Jose F. Moran", "Gautam Sarath" ], "abstract": "Hemoglobins (Hbs) corresponding to non-symbiotic (nsHb) and truncated (tHb) Hbs have been identified in rice (Oryza). This review discusses the major findings from the current studies on rice Hbs. At the molecular level, a family of the nshb genes, consisting of hb1, hb2, hb3, hb4 and hb5, and a single copy of the thb gene exist in Oryza sativa var. indica and O. sativa var. japonica, Hb transcripts coexist in rice organs and Hb polypeptides exist in rice embryonic and vegetative organs and in the cytoplasm of differentiating cells. At the structural level, the crystal structure of rice Hb1 has been elucidated, and the structures of the other rice Hbs have been modeled. Kinetic analysis indicated that rice Hb1 and 2, and possibly rice Hb3 and 4, exhibit an extremely high affinity for O2, whereas rice Hb5 and tHb possibly exhibit a low to moderate affinity for O2. Based on the accumulated information on the properties of rice Hbs and data from the analysis of other plant and non-plant Hbs, it is likely that Hbs play a variety of roles in rice organs, including O2-transport, O2-sensing, NO-scavenging and redox-signaling. From an evolutionary perspective, an outline for the evolution of rice Hbs is available. Rice nshb and thb genes vertically evolved through different lineages, rice nsHbs evolved into clade I and clade II lineages and rice nshbs and thbs evolved under the effect of neutral selection. This review also reveals lacunae in our ability to completely understand rice Hbs. Primary lacunae are the absence of experimental information about the precise functions of rice Hbs, the properties of modeled rice Hbs and the cis-elements and trans-acting factors that regulate the expression of rice hb genes, and the partial understanding of the evolution of rice Hbs.", "keywords": [ "Evolution", "function", "gene expression", "non-symbiotic", "structure", "symbiotic", "truncated" ], "content": "Abbreviations\n\n2,4-D, 2,4-dichlorophenoxyacetic acid; ARR1, Arabidopsis response regulator 1; BCIP, 5-bromo-4-chloro-3´-indolyphosphate; Hb, hemoglobin; Lb, leghemoglobin; MIP1, macrophage inflammatory protein 1; mya, million of years ago; NBT, nitro-blue tetrazolium; nsHb, non-symbiotic hemoglobin; nsHb-1, non-symbiotic hemoglobin type 1; nsHb-2, non-symbiotic hemoglobin type 2; nsHb-I, clade I non-symbiotic hemoglobin; nsHb-II, clade II non-symbiotic hemoglobin; RT-PCR, reverse transcriptase-polymerase chain reaction; SNP, sodium nitroprusside; tHb, truncated (2/2) hemoglobin.\n\n\nIntroduction\n\nTwo decades ago Taylor and co-workers reported the cloning and sequencing of a hemoglobin (Hb) cDNA from barley1. This was the first report about the existence of Hbs in monocotyledonous plants. Since then Hbs have been identified in a number of monocots, including rice2, maize3 and wheat4. Rice Hbs and genes coding for these proteins are well characterized, thus in many aspects rice Hbs are a model to understand monocot and other land plant Hbs. This review discusses major findings from the study of rice Hbs including a historical perspective, and proposes biochemical and physiological mechanisms for rice Hbs based on information available about rice Hbs and other monocot and land plant Hbs. For general aspects and the biochemistry, physiology and evolution of plant Hbs, we recommend to the reader reviews published elsewhere5–18.\n\nHb is known to the reader because this protein is responsible for the red color of vertebrates’ blood19. However, Hbs are widely distributed in living organisms, ranging from bacteria to mammals20–22. The tertiary structure of Hbs consists of a specific arrangement of 6 to 8 α-helices (designated with letters A to H) known as the globin-fold. This protein folding forms a hydrophobic pocket where a Fe-heme prosthetic group is located19,23,24. Two structural types of the globin-fold have been identified in Hbs: the 2/2- and 3/3-folding. In the 2/2-Hbs, helices B and E overlap to helices G and H and in the 3/3-Hbs helices A, E and F overlap to helices B, G and H. Likewise, three evolutionary families have been identified in Hbs: the M, S and T Hb families. The M Hbs, which exist in bacteria and eukaryotes, include flavoHbs and single domain globins, the S Hbs, which exist in bacteria and yeasts, include globin-coupled sensors, protoglobins and single domain globin sensors, and the T Hbs, which exist in bacteria, unicellular eukaryotes and plants, include truncated Hbs (tHbs). Canonical T Hbs from bacteria and unicellular eukaryotes are ~100 to 120 amino acids in length, however plant T Hbs are longer than canonical T Hbs because of the existence of extra amino acids at the N- and C-terminal. The M and S Hbs fold into the 3/3-folding whereas the T Hbs fold into the 2/2-folding21,25–28.\n\nA variety of ligands bind to the Fe-heme of Hbs, including O2 and NO. Reversible binding of O2 is closely associated to the major function of Hbs in organisms, which is the transport of O219. Binding of NO by Hbs is essential to NO-detoxification via a NO-dioxygenase activity29. Several additional functions have been reported for Hbs, including dehaloperoxidase activity and reaction with free radicals, binding and transport of sulfide and lipids, and O2-sensing30–39. This indicates that in vivo Hbs might be multifunctional proteins.\n\nLand plant Hbs were first identified by Kubo in soybean root nodules40. Few years after Kubo’s discovery these proteins were named as leghemoglobins (Lbs) by Virtanen and Lane41 because they were only found in the symbiotic (N2-fixing-) nodules of the leguminous plants. Lbs are the most abundant soluble proteins in nodules (e.g. in soybean nodules their concentration is as high as 3 mM)16,42. The x-ray analysis of lupin Lb revealed that the tertiary structure of Lbs was remarkably similar to that of the sperm whale myoglobin43. This evidence demonstrated that Lbs are plant Hbs and indicated that plant and animal Hbs evolved from a common ancestor more than 600 mya6. Subsequent work led to the identification of Lb-like (or symbiotic) Hbs in nodules of actinorhizal plants44–48, purification of an Hb from the root nodules of the dicotyledonous non-legume Parasponia andersonii49, cloning and sequencing of an hb gene from the non-nodulating dicot Trema tomentosa50,51 and detection of Hbs in non-symbiotic organs from several land plants, including primitive bryophytes and evolved angiosperms9,18,52–54. Until now three types of Hbs have been identified in land plants: the symbiotic Hbs, which include Lbs, that are specifically located within nodules of the N2-fixing land plants, and the non-symbiotic (nsHbs) and truncated (tHbs) Hbs, that are located within non-symbiotic and symbiotic organs of primitive and evolved land plants9,18. Based on sequence similarity the nsHbs are further classified into type 1 and type 2 nsHbs (nsHbs-1 and nsHbs-2, respectively)9,17,55.\n\nMonocots are a large family of flowering plants56–58 that includes cereals. Cereals, such as rice, maize and wheat, are the main source of food for humans59. Because of this, during that last decade the genomes of a number of cereals have been sequenced. This allowed the identification of novel cereal Hbs. The search of hb genes in databases by G. Rodríguez-Alonso and R. Arredondo-Peter60,61 revealed that nsHb and tHb sequences exist in the Brachypodium distachyon, Hordeum vulgare (barley), Oryza glaberrima (rice), O. rufipogon (rice), O. sativa (rice) var. indica, O. sativa (rice) var. japonica, Panicum virgatum (switchgrass), Setaria italica (foxtail millet), Sorghum bicolor (sorghum), Triticum aestivum (wheat) and Zea mays ssp. mays (maize) genomes. The highest number of nsHbs (5) exists in O. sativa var. indica and O. sativa var. japonica, whereas one to three nsHbs exist in barley, Brachypodium, foxtail millet, maize, O. glaberrima, O. rufipogon, sorghum, switchgrass and wheat. Also, with the exception of wheat, which contains two copies of the thb gene, a single copy of thb was identified in the genome of Brachypodium, barley, O. sativa var. indica, O. sativa var. japonica, switchgrass, foxtail millet, sorghum and maize. Little is known about Hbs from non-cultivated monocots. The only Hb reported from a non-cultivated monocot is that of teosinte (Z. mays ssp. parviglumis)3, which is postulated as the ancestor of maize59,62–67. Analysis by Southern blot using the teosinte hb gene as probe showed that apparently a single copy of hb exists in teosinte (J. Sáenz-Rivera and R. Arredondo-Peter, unpublished results). Sequence comparison revealed that maize and teosinte Hb polypeptides are identical3.\n\nMonocots were a target for searching Hbs after these proteins were detected in non-symbiotic organs of dicotyledonous plants (see subsection above). At that time, monocot genomes had not been sequenced. Searching approaches consisted in detecting Hb polypeptides and hb genes by spectroscopy and molecular biology methods, respectively. Attempts to detect absorption maxima in the Søret (~410 nm) and Q (~500 to 550 nm) regions, which are characteristic of ferric (Fe3+), ferrous (Fe2+) and liganded Hbs68,69, were unsuccessful (R. V. Klucas and C. A. Appleby, unpublished results) mostly due to the very low Hb concentration (~50 to 100 nM) in plant non-symbiotic organs5,70. At the molecular level a consensus probe designed from legume and non-legume (T. tomentosa, P. andersonii and Casuarina glauca) Hb sequences71 hybridized with hb-like sequences from rice and other monocot total DNAs (Figure 1). This observation suggested that hb sequences exist in monocots, however hybridizing fragments were not subsequently cloned and sequenced in order to verify if they actually corresponded to hb genes.\n\nApproximately 20 μg of undigested total DNA was used as template and a consensus oligonucleotide for legume and non-legume plant Hbs71 was used as probe. Sequence of the consensus probe was 5´-GTA GCC TAT GAT GAA TTG GCA GCT GCA ATT AAG-3´. The probe was labeled by nick translation with Biotin-dATP using a Bionick labeling system (Gibco BRL). The membrane was prehybridized with SSC 2× for 4h at 42°C, hybridized overnight at the same temperature, washed at high stringency (SSC 2×/SDS 0.1% for 3 min at room temperature, SSC 0.2×/SDS 0.1% for 15 min at room temperature and SSC 0.16×/SDS 0.1% for 15 min at 65°C) and incubated with the streptavidin-alkaline phosphatase conjugate and the BCIP/NBT mix to develop color. Animal (salmon sperm and calf thymus) and legume DNAs were included as negative and positive controls, respectively.\n\nRice Expressed Sequence Tags (ESTs) were first deposited in databases early in the 1990’s. The first rice Hb (Hb1 and Hb2) sequences were detected from ESTs deposited in the DNA Data Bank of Japan (DDBJ) database72. Rice Hb1 and Hb2 corresponded to clones C741 and C2576 with DDBJ accession number D15507 and D38931, respectively. Rice hb1 and hb2 genes were subsequently amplified by PCR, cloned and sequenced. Sequence analysis revealed that rice hb1 codes for non-symbiotic Hb1 and that rice hb2 codes for non-symbiotic Hb22. Afterwards, sequencing of the rice (O. sativa L. ssp. indica) genome more than a decade ago73 allowed the identification of a family of rice nshb genes and a single copy of the rice thb gene (see subsection below).\n\n\nMolecular biology of rice hemoglobins\n\nThe O. sativa var. indica and O. sativa var. japonica genomes are fully sequenced, and the O. glaberrima and O. rufipogon genomes are partially sequenced. Rice genome sequences are mainly available from the GenBank (www.ncbi.nlm.nih.gov) and Phytozome (http://www.phytozome.org/) databases. Search of Hb sequences in the above databases showed that a family of the nshb genes, consisting of hb1, hb2, hb3, hb4 and hb5, and a single copy of the thb gene exist in the O. sativa var. indica and O. sativa var. japonica genomes. A single copy of the nshb gene was detected in the O. glaberrima and O. rufipogon genomes, however thb genes have not yet been detected in these plants61. Given that the sequencing of the O. glaberrima and O. rufipogon genomes is in progress the identification of hb genes in these genomes is incomplete. Thus, the following discussion will focus on the O. sativa var. indica and O. sativa var. japonica hbs.\n\nThe structure of known rice hb genes corresponds to four exons and three introns, with introns located at similar position as all of the known plant hb genes74. Canonical TATA boxes and a variety of potential promoters exist upstream of the rice hb genes which suggests that rice hbs are functional and that the regulation of the hb genes in this plant is complex75–77. Figure 2 shows the localization of hbs in the O. sativa chromosomes and mapping of hbs in the O. sativa genome. Rice hb1, hb3 and hb4 cluster forming the hb1-hb4 cluster76 which is localized in chromosome 3. Rice hb2 is also localized in chromosome 3 but 467 kb upstream of the hb1-hb4 cluster. In contrast, rice hb5 and thb genes are localized in chromosomes 5 and 6, respectively (Figure 2A). Rice hbs are flanked by a variety of genes with known and unidentified functions (Figure 2B). However, with the exception of genes coding for a ternary complex factor macrophage inflammatory protein MIP1 and an ubiquitin fusion protein which are located 239 and 411 nucleotides up- and downstream of the hb1-hb4 cluster, respectively, distance of flanking genes to hbs is >1 kb. This suggests that co-expression of hb and flanking genes is unlikely.\n\nThe hb genes were localized in the rice chromosomes by BlastN analysis using the rice (O. sativa) genome resource from the GenBank database as template and the sequence for the rice hb1, 3 and 4 (GenBank accession number AF335504), hb2 and 5 (GenBank accession numbers AF335503 and EF061459, respectively) and thb (GenBank accession number NM_001064507) genes as probes. The hb (black boxes) and flanking (gray boxes) genes were mapped into 50 kb fragments of the O. sativa genome by BlastN2.2.26+ analysis using the Phytozome V9.1 server (www.phytozome.org) and the above hb sequences as probes. Arrows indicate the transcription orientation. Information for each gene corresponds to predicted protein (following the Phytozome nomenclature), locus name in the O. sativa genome and position at the O. sativa chromosome. Gene sizes and distance between genes are not shown at scale. Pltd, chloroplast chromosome; MT, mitochondrial chromosome.\n\nThe expression of hb genes and localization of Hb polypeptides have been analyzed in rice growing under normal and stressed conditions. Under normal conditions the expression level of rice nshbs was low2,75. However, analysis by RT-PCR revealed that hb1, hb2 and hb5 genes were expressed in embryonic and vegetative organs obtained from rice plants grown under a normal environment2,75,78. Specifically, transcripts for rice Hb1 were detected in embryos, seminal roots, leaves and roots, transcripts for rice Hb2 were detected in embryos, coleoptiles, seminal roots and leaves, and transcripts for rice Hb5 were detected in embryos, coleoptiles, seminal roots, leaves and roots. Likewise, evaluation of the β-glucuronidase (GUS) activity from a construct containing the rice nshb2 gene promoter that is responsive to the cytokinin-regulated ARR1 trans-acting factor showed that this promoter is activated in roots, the vasculature of young leaves, flowers and the pedicel/stem junction of transgenic Arabidopsis77. In addition, a variety of potential promoters was identified upstream of the rice nshb genes, such as those involved in the ethylene synthesis, photoregulation, heat shock response and plant defense signaling70,75–77. However the activities of these promoters have not been determined.\n\nTranscriptomic analyses revealed that nsHb and tHb transcripts coexist in rice embryonic and vegetative organs (Table 1). This evidence suggests that nsHb (i.e. Hb1, Hb2, Hb3, Hb4 and Hb5) and tHb polypeptides coexist and probably function in rice organs. Immunoanalysis by Western blot and confocal microscopy using a polyclonal anti-rice Hb1 antibody revealed that Hb polypeptides exist in rice seeds and in rice leaves and roots from 2 to 14 weeks after seed germination. These analyses also revealed that Hb polypeptides exist in the cytoplasm of differentiating cells of the root cap, schlerenchyma, aleurone, and in the vasculature, principally in the differentiating xylem16,70,79. However, the anti-rice Hb1 antibodies cross-react with different rice Hbs (G. Sarath and E. J. H. Ross, unpublished results) and thus it is not known which Hb polypeptides were detected in the above analyses by the anti-rice Hb1 antibodies.\n\nRice Hb transcripts were detected in plant organs using The Rice Genome Annotation Project database (http://rice.plantbiology.msu.edu/) and hemoglobin as keyword (S. Castro-Bustos and R. Arredondo-Peter, unpublished).\n\nIt is well documented that land plant hb genes are either up- or down-regulated by stress conditions1,52,54,79–82. Table 1 shows that Hb transcripts coexist in rice growing under cold, drought and salt stress conditions. Also, Ohwaki and co-workers83 reported that nshb1 and nshb2 are induced by nitrate, nitrite and NO in cultured rice cells. These observations indicate that rice hb genes response to a variety of stress conditions. However, the detection of Hb polypeptides by Western blot using the anti-rice Hb1 antibodies showed that level of Hbs increased in rice etiolated leaves and flooded roots, but not in rice plants subjected to oxidative (H2O2), nitrosative (SNP) and hormonal (2,4-D) stresses. These observations suggest that rice Hbs do not appear to be part of a generalized stress response, but may be functional in plant organs subjected to specific stress conditions79.\n\n\nStructure and biophysical properties of rice hemoglobins\n\nRice hb genes are functional and code for Hb polypeptides with a predicted molecular mass of ~16 to 19 kDa2,74–76. Rice Hb1 was the first monocot nsHb whose crystal structure was elucidated84. This protein crystalizes as a dimer, thus it is possible that in vivo rice Hb1 forms dimers when its concentration is ≥1 mM85. After the elucidation of the rice Hb1 structure the tertiary structure of rice Hb286, Hb3, Hb4 and Hb575 (CASPUR PMDB ID PM0075009, PM0075873, PM0076005 and PM0075011, respectively) was predicted using computational methods and rice Hb1 (PDB ID 1D8U) as the structural homolog. The crystal structure of rice Hb1 and that of predicted rice Hb2, Hb3 and Hb4 is highly similar. The tertiary structure of these proteins consists of six helices that fold into the 3/3-folding (see subsection on Generalities on hemoglobins). However, the structure of rice Hb1 to 4 is characterized by the existence of a short pre-helix A located at the N-terminal and an extended and poorly ordered CD-loop. The heme pocket in these proteins differs from that in “traditional” Hbs because the proximal and distal His side chains coordinate the Fe-heme forming a hemichrome (Figure 3), resulting in that Fe-heme from rice Hb1 to 4 is hexacoordinate. Also, the amino acid residues (V50, S53, E123, V124, F127 and A128) located at the monomer-monomer interface of dimeric rice Hb184 are highly conserved in rice Hb2 to 476. This suggests that rice Hb1 to 4 can potentially form homo- or hetero-dimers if the hb1 to 4 genes coexpress in rice organs. The tertiary structure of rice Hb5 also consists of six helices that fold into the 3/3-folding. However, rice Hb5 differs from rice Hb1 to 4 in missing 11 amino acids in helix E which results in that the length of the CD-loop and helix E in the predicted Hb5 structure are unusually long and short, respectively. An apparent consequence from this characteristic is that distal His is located far away (13.92 Å, compared to 2.11 Å in rice Hb1) from the Fe-heme within the predicted Hb5 structure, resulting in that Fe-heme from rice Hb5 could be pentacoordinate75. The amino acid residues located at the monomer-monomer interface of dimeric rice Hb184 are poorly conserved in rice Hb575 which suggests that rice Hb5 exists in vivo as a monomer.\n\nThe tertiary structure of rice tHb was modeled using the automated mode of the I-Tasser server (http://zhanglab.ccmb.med.umich.edu/I-TASSER/)153–155 and the crystal structure of the Thermobifida fusca tHb (PDB ID 2BMM) as the structural homologue. Model for the rice tHb is deposited in the Caspur Protein Model Database (http://bioinformatics.cineca.it/PMDB/main.php) under the ID number PM0079484. Helices are indicated with letters A to H. Note the overlapping of helices A, E and F to helices B, G and H in (3/3-folding) rice Hb1, and overlapping of helices B and E to helices G and H in (2/2-folding) rice tHb. Heme prosthetic group is shown in dark green color and proximal and distal His are shown in light brown color.\n\nThe folding pathway and kinetics of rice nsHbs were predicted using the Average Distance Map (ADM) method87–89. This analysis indicated that rice Hb1 and Hb2 could fold in the C → N direction at a moderate rate, that rice Hb3 could fold in the N → C direction at a fast rate, and that rice Hb4 and Hb5 could fold in the N → C direction at a moderate rate. Thus, it appears that the predicted folding pathway and kinetics among rice nsHbs are diverse. Also, the ADM analysis showed that pre-helix A and CD-loop apparently do not play a role during the folding of rice nsHbs90. The ADM analysis has not been performed on rice tHb, thus the predicted folding pathway and kinetics for this protein are not known.\n\nRice (O. sativa) tHb (GenBank accession number NP_001057972) is 172 amino acids in length, which corresponds to a globin domain (position 26 to 147) flanked by N- and C-terminal extensions. No monocot tHb has been analyzed by x-ray crystallography, however the tertiary structure of a rice tHb was predicted using computational methods (Figure 3). The predicted structure of rice tHb is highly similar to the crystal structure of an Arabidopsis thaliana tHb91. The globin domain from rice and A. thaliana tHbs folds into the 2/2-folding (see subsection on Generalities on hemoglobins). Similarly to the A. thaliana tHb structure, flanking regions to the globin domain of predicted rice tHb correspond to an N-terminal helical extension and a C-terminal unfolded extension (Figure 3). The high similarity between the crystal structure of A. thaliana tHb and the predicted structure of rice tHb suggests that the biochemical properties and function of dicot and monocot tHbs are similar.\n\nVisible spectroscopy (see subsection Early search and identification of rice hemoglobins) is a tool to analyze the redox state of and ligand-binding to the Fe-heme of Hbs44,68,69,92,93. Rice Hb1 is the only rice nsHb that has been spectroscopically characterized2. This protein exhibits spectral characteristics that are similar to other Hbs. However, rice Hb1 exhibits distinctive absorption maxima in the deoxyferrous form: the unligated ferrous state exhibits maxima at 526 and 556 nm2 which are characteristic of hexacoordinate Fe-heme94. This is in contrast to pentacoordinate Hbs which display a broad peak centered at 556 nm in their deoxyferrous form7,95,96. The distal ligand that coordinates the Fe-heme in rice Hb1 was identified as His74 by site directed mutagenesis. Absorbance spectra of the ferric and deoxyferrous forms of an H74L mutant of rice Hb1 showed no evidence of His coordination. Also, the addition of exogenous imidazole to ferric and deoxyferrous H74L mutant resulted in a spectrum identical to that of the wild-type rice Hb12. This evidence indicated that in rice Hb1 the distal ligand to Fe-heme is His74. A similar case can be predicted for rice Hb2 to 4. In contrast, distal His appears to be located far away from the Fe-heme in the predicted structure of deoxyferrous rice Hb5, resulting in that Fe-heme in rice Hb5 could be pentacoordinate75.\n\nRice tHb has not been subjected to spectral analysis, however the predicted structure of this protein (Figure 3) is highly similar to the crystal structure of an A. thaliana tHb91 (see subsection on Structure of rice hemoglobins). The absorption spectra of an A. thaliana tHb showed that Fe-heme from this protein is pentacoordinate82,91. Thus, it is likely that Fe-heme in rice tHb is pentacoordinate and that the O2-binding properties of rice tHb are similar to those of pentacoordante Hbs, i.e. the O2-association and -dissociation rate constants are high.\n\nAnalysis of ligand-association and -dissociation rate constants of penta- and hexacoordinate Hbs using stopped-flow methods indicated that these proteins exhibit low to moderate and high affinity for O2, respectively. Rice Hb1 to 4 are hexacoordinate and apparently rice Hb5 and tHb are pentacoordinate. The O2-association rate constants for hexacoordinate rice Hb1 and 2, and possibly for rice Hb3 and 4, are similar to those of other O2-transport and -storage proteins, such as the spermwhale myoglobin and soybean Lba8,16,75,97. However, in rice Hb12 and 216, and possibly in rice Hb3 and 4, the bound O2 is stabilized by distal His after binding to the Fe-heme, which results in very low O2-dissociation rate constants. The O2-association and -dissociation rate constants of hexacoordinate rice Hb1 and 2, and possibly of rice Hb3 and 4, result in that the affinity of these proteins for O2 is extremely high (e.g. KO2 = 1,800 and 1,316 μM-1 for rice Hb1 and 2, respectively2,16). In contrast, the O2-association and -dissociation rate constants of pentacoordinate rice Hb5 and tHb could be moderate to high, which could result in a low to moderate affinity for O2.\n\nThe bis-histidyl hexacoordinated form of rice Hb1 displays a hydrophobic distal cavity which appears to be connected with the external solvent through the position of Phe44 (also known as FB10 because it occupies the tenth position in helix B). It was suggested that this amino acid regulates the migration of small ligands in rice Hb1, for example in ligand binding to the Fe-heme, ligand migration through internal docking sites and ligand release into the external solvent98,99. Kinetic analysis after laser flash photolysis of rice Hb1 encapsulated in silica gel combined with computational analysis revealed the existence of two channels in the rice Hb1 CO-bound species. The first channel is located in the distal region of the heme pocket and is connected with a secondary channel that is directly connected with the external solvent. Apparently, the position of FB10 in hexacoordinated rice Hb1 leaves the distal heme pocket accessible to the external solvent, however after the ligand entrance the phenyl ring rotates closing the cavity and thus hindering the exit of the bound ligand100. Thus, together with distal (H74) His (see subsection Spectroscopic characteristics of rice hemoglobins) and aromatic (F40, F41, F55, F57, Y145 and L126) amino acids that are located in the distal region of the heme pocket, FB10 appears to regulate hexacoordination and functioning of rice Hb1.\n\n\nPostulated functions for rice hemoglobins\n\nWhile data on the localization, kinetics, regulation and structure of rice Hbs have accumulated, little work has been performed to fully understand the function of these proteins in rice organs. However, previous work from other plant and non-plant Hbs provides data that enable us to propose potential functions for rice Hbs. Rice Hbs could potentially function within cells through O2-transport and -signaling, binding to small molecules (most notably NO) and other as yet undetermined mechanisms. Here we evaluate the evidence for and against these modes of action.\n\nOxygen transport is a major function of many Hbs. This process requires that the kinetics of O2-binding do not limit the O2-diffusion process101–104. Based on the concentration of Hb polypeptides in rice organs (~50 to 100 nM)70, the O2-association rate constant of rice Hb1 and 2 (68 and 50 μM-1 s-1, respectively)2,16 and possibly that of rice Hb3 to 5 and tHb (see subsection Kinetic properties of rice hemoglobins), and the free O2 concentration in aerated rice roots (<1.4 μM)105, it is likely that Hbs would be substantially oxygenated in rice organs. However, the O2-dissociation rate constants of rice Hb1 and 2 (kO2 = 0.038 s-1 2,16), and possibly that of rice Hb3 and 4, are extremely low. These data do not support the O2-transport function for rice Hb1 to 4 because these proteins would not release O2 after oxygenation. In contrast, the predicted kinetic constants for rice Hb5 and tHb suggest that these proteins bind and release O2 easily and thus that they function by transporting O2.\n\nIt was reported that hexacoordinate Hbs interact with either organic molecules or protein partners31,38 and thus a possibility is that such interactions could impact the kinetic constants, particularly the O2-dissociation rate constants, of hexacoordinate nsHbs106. There have been no direct biochemical evaluations of this hypothesis in rice or in other plants, precluding definitive answers. However, their unique structural features could result in as yet undiscovered interactions.\n\nRice Hbs may function in O2-signaling if they easily bind and release O2. Appleby and co-workers5 proposed that under normal conditions Hbs would be oxygenated and under O2-limiting conditions the concentration of deoxyHb would increase triggering an anaerobic response. It was reported that levels of Hbs increase in rice roots from flooded plants indicating that the synthesis of rice Hbs increases under O2-limiting conditions79. Rice is a flooding resistant crop, thus under flooding (i.e. hypoxia) conditions rice Hbs could sense low O2-concentrations and trigger an anaerobic metabolism for rice growth. To act as a signaling molecule, rice Hbs will need to bind directly to the DNA, to additional proteins, such as transcription factors, or catalyze some unique reactions that can influence key downstream events. To date there are no reports of immunoprecipitation experiments specially targeting rice Hbs coupled to further proteomic analysis. It is thus uncertain if rice Hbs bind to other partners. There is also no structural evidence that indicates that rice Hbs can bind directly to DNA. In planta, they appear to be soluble and essentially contained within the cytoplasm70. There are reports of nuclear-localized Hbs107, but no direct evidence for a function arising from translocation of Hbs from the cytoplasm to the nucleus currently exist.\n\nThe NO dioxygenase activity exhibited by Hbs is well documented108–110. NO is a hormone-like radical that modulates several aspects of the plant physiology, including plant immunity, seed germination, de-etiolation, apoptosis, stomata guard cells opening/closure and the rhizobia-legume symbiosis111–122. Scavenging of NO is considered a function of plant Hbs11,123–126. During this process, oxygenated plant Hbs react with NO producing nitrate and oxidizing ferrous Hb to the ferric form. Ferric plant Hbs are subsequently reduced to ferrous Hb by enzymatic127,128 and non-enzymatic129–133 mechanisms. This process regenerates ferrous Hb which is able to bind NO in a cyclic pathway referred to as the Hb/NO cycle126,134. The operation of this cycle appears to be involved in maintaining an active metabolism in the plant cells11. Rice Hb1 exhibits NO dioxygenase activity (kobs, NOD = 90 s-1)135 thus a possible function of Hbs into the rice physiology is modulating levels of NO by scavenging NO. However, the inability of rice Hb1 to substitute the NO scavenger activity in a flavoHb knockout Escherichia coli135 and the observation that levels of Hbs did not change in rice seeds germinated under nitrosative stress79 suggest that the NO dioxygenase activity of rice Hbs is limited in vivo.\n\nA consequence of the operation of the Hb/NO cycle could be the maintenance of cell respiration and energy status. Based on the studies on over- and under-expressing barley nsHb in maize cells, it was proposed that under hypoxic conditions barley nsHb is involved in the ATP metabolism, particularly in maintaining the energy status under O2-limiting conditions136. Immunolocalization data showed that rice Hbs are localized in differentiating cells (see subsection on Gene expression and localization of hemoglobins in rice organs)70. The metabolism of these cells is redirected in response to differentiation signals, such as a change in the cell redox state. Rice Hbs could be involved in redox signaling if the redox state of the heme is functional84. Thus, under these conditions rice Hbs may function by sensing or maintaining redox environments that promote specific cell metabolisms16.\n\nIt was proposed that one of the functions of plant Hbs could be related to the peroxidase activity8,106. This is of interest because peroxidase activity modulates the levels of reactive oxygen species and a variety of cellular processes137–143. In plants, evaluation of the peroxidase activities of Arabidopsis Hbs (AtGLB1, AtGLB2 and AtGLB3) revealed that these proteins oxidize Amplex Red, DHR123 and guaiacol substrates144 and overexpression of AtGLB1 increased tolerance of Arabidopsis to H2O2 stress145. These observations suggested that Arabidopsis Hbs function as antoxidants. However, levels of Hb polypeptides did not change in rice seeds germinated under H2O2 stress79. Also, the analysis of the peroxidase activity of rice Hb1 compared to that from horseradish peroxidase (HRP) showed that the catalytic efficiency of rice Hb1 for the oxidation of guaiacol using H2O2 as electron donor is several orders of magnitude lower than that of HRP (kcat/Km = 15.8 and 44,833 mM-1min-1, respectively). Additionally, it was observed that recombinant rice Hb1 poorly protects E. coli from H2O2 stress146. This evidence indicates that it is unlikely that rice Hbs function in vivo as peroxidases.\n\nBased on gene expression (Table 1), protein localization and structural and kinetic properties of rice Hbs and data from the analysis of other plant and non-plant Hbs it is likely that Hbs play a variety of roles in rice plants growing under normal and stressed conditions. These functions may include O2-transport, O2-sensing, NO-scavenging and redox-signaling. Future work on rice Hbs should focus on testing the above potential functions as well as newly proposed functions that emerge from novel observations.\n\n\nEvolution of rice hemoglobins\n\nHbs are widely distributed in land plants, ranging from primitive bryophytes to evolved angiosperms9. The outline of plant Hb evolution subsequent to land colonization was clarified18. Briefly, a phylogenetic analysis showed that plant and animal hb genes diverged 900-1,400 mya, that land plant nshb and thb genes vertically evolved through different lineages from algal ancestors, that nsHbs-1 and nsHbs-2 are monophyletic and evolved via a gene duplication event prior to the divergence of monocots and dicots at ca. 140 mya, and that symbiotic hbs originated from nshb genes at ca. 94 mya. Likewise, the structural analysis of primitive nsHbs and Lbs revealed that changes during the evolution of nsHbs to Lbs were a hexacoordinate to pentacoordinate transition at the heme prosthetic group, a length decrease at the CD-loop and N- and C-terminal regions, and a compaction of the protein into a globular structure54,147.\n\nIn contrast, the evolution of rice Hbs is partially understood owing to the limited availability of Hb sequences from a wide variety of wild and cultivated rice. However, the outline of monocot Hb evolution is rather well understood. Thus, in this section we will discuss the evolution of rice Hbs within the context of major events that occurred during the evolution of monocot Hbs. A major event during the evolution of land plant nsHbs was the duplication of an ancestral nshb into nshb-1 and nshb-2 prior to the monocot-dicot divergence18,148. Sequence analysis revealed that nshb-1 and nshb-2 genes exist in dicots and that apparently only nshb-1 genes exist in monocots9,94,149. Earlier Garrocho-Villegas and co-workers75 reported the existence of a nsHb (Hb5) divergent from rice (Hb1 to 4) nsHbs-1 and suggested that nsHbs divergent from nsHbs-1 evolved within monocots. Subsequent phylogenetic analysis of monocot nsHb sequences revealed that apparently only nshb-1 evolved within monocots, that nshb-1 duplicated early in the evolution of monocots originating clade I and clade II nshbs (nshbs-I and nshbs-II, respectively), that nsHbs-I correspond to dicot nsHbs-1, and that nsHbs-II diversified into regular nsHbs-II, post-helix H-containing nsHbs-II and 11 amino acids deletion-containing nsHbs-II60. This analysis also showed that O. sativa var. indica and O. sativa var. japonica Hb1 to 4 and Hb5 cluster within clade I and clade II, respectively, and that O. glaberrima and O. rufipogon (whose all nshb copies remain unidentified because their genome sequencing is in progress) nsHbs cluster within clade I. Thus, apparently clade I and clade II lineages remain conserved during the evolution of rice nsHbs60.\n\nEvaluation of the rate of divergence of selected land plant Hbs revealed that evolutionary rates slowed down previous to the origin of magnoliophyta and that the rate of divergence was slower in rice Hb1 than in rice tHb150. This observation suggested that rice Hb1 (and conceivably other rice nsHbs) evolved under the effect of the stabilizing selection. However, the estimation of the variability of the O. sativa var. indica, O. sativa var. japonica, O. glaberrima and O. rufipogon nshb and thb genes revealed that in these plants variability is higher in nshbs than in thbs and that these genes evolved under the effect of neutral selection61. Currently the effect of rates of divergence and gene variability on the Hbs function during the rice evolution is not known.\n\n\nConcluding remarks and future directions\n\nIn the preceding sections of this review we summarized major findings from the study of rice Hbs. This review also reveals some major lacunae in our ability to completely understand rice Hbs, more specifically the lack of information about the precise functions of Hbs in rice organs. The proposed functions for rice Hbs are mostly based on the analysis of other plant and non-plant Hbs. Thus, future work should evaluate the Hb activities (e.g. the NO-binding and -detoxifying activities) in either rice organs or rice cell cultures under a variety of growing conditions. Elucidating the functions of rice Hbs also requires the identification of either homo- or heterodimeric rice Hbs and possible organic molecules and protein partners that interact with rice Hbs. Other lacunae are the absence of biochemical, biophysical and cellular data on the properties of rice Hb2 to 5 and tHb. Generating recombinant rice Hb2 to 5 and tHb should provide Hb polypeptides for a variety of analyses that reveal the biochemical and biophysical properties of these proteins.\n\nWith the exception of rice hb2, a lacuna is the absence of experimental information about the cis-elements and trans-acting factors that regulate the expression of rice hbs. This information may help to integrate the hb gene expression into the rice metabolisms, including those that are modulated by plant hormones.\n\nA final lacuna is the incomplete understanding of the evolution of rice Hbs. Sequencing of the O. glaberrima and O. rufipogon genomes will be completed soon and most likely a number of rice genomes (including that of O. barthii, which is postulated as the ancestor of O. glaberrima59,151,152) will be sequenced within the near future. This will provide new Hb sequences for phylogenetic analysis and the understanding of the evolution of rice Hbs, including the identification of ancestral rice Hbs and the evaluation of the effect of rice domestication and breeding during the evolution of rice Hbs.", "appendix": "Author contributions\n\n\n\nRAP conceived the review and prepared the first draft of the manuscript. RAP, JFM and GS were involved in the revision of the draft manuscript and prepared the final version.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nTaylor ER, Nie XZ, MacGregor AW, et al.: A cereal haemoglobin gene is expressed in seed and root tissues under anaerobic conditions. Plant Mol Biol. 1994; 24(6): 853–862. PubMed Abstract \| Publisher Full Text\n\nArredondo-Peter R, Hargrove MS, Sarath G, et al.: Rice hemoglobins. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "6520", "date": "13 Nov 2014", "name": "Martino Bolognesi", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe Review by Arredondo-Peter et al. presents our current knowledge on the fascinating field of plant non-symbiotic (nsHb) and truncated hemoglobins (tHb), for which substantial, but scattered, information has accumulated over the past twenty years. The Review deals specifically with rice Hbs. Work from several distinct worldwide groups has so far provided information on the gene families for five nsHbs, and for a single tHb in rice; moreover, expressed proteins have been located to various plant organs and developmental stages. Crystal structures and kinetic analyses have helped delineating the potential roles of rice Hbs in plant physiology, highlighting different O2 binding affinities that differentiate the various Hbs. A main question that remains unanswered concerns the in vivo functions carried over by the five distinct rice nsHbs and tHb; hints reviewed from the literature include O2 sensing, signaling, O2 transport, NO scavenging, and NO dioxygenase pseudo-enzymatic activities (others may also be plausible). The Review includes evolutionary considerations (and the effects of rice selection through domestication) that will be reinforced by the forthcoming completion of rice genomes.Overall, the Review provides a useful compendium over a subject whose reunification into a coherent presentation will indeed support deeper exploration of the functional aspects, whose scientific and practical relevance cannot be underestimated.", "responses": [ { "c_id": "1083", "date": "16 Nov 2014", "name": "Raul Arredondo-Peter", "role": "Author Response", "response": "We thank Dr. Bolognesi for evaluating this review and his comments." } ] }, { "id": "6522", "date": "20 Nov 2014", "name": "Juliette T. J. Lecomte", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript “Rice (Oryza) hemoglobins” by Arredondo-Peter, Moran and Sarath reviews the current status of research on the group of proteins found within rice plants belonging to the hemoglobin (Hb) superfamily. There is growing interest in Hbs, particularly non-mammalian Hbs, owing to their potential role as mediators of reactive nitrogen molecules, such as nitric oxide. Such a review is welcome, as rice is an important cultivar and adaptation to stress during growth plays an important role in the survival of the plant. The review covers a wide range of relevant topics, such as phylogenetic aspects, cellular localization, and chemical properties. Within this review, the literature on rice Hbs is covered thoroughly, as witnessed by the extensive bibliography that accompanies the manuscript. However, closer inspection of that bibliography reveals an underlying issue with this manuscript. Although over 150 citations are given, a minority of those papers reference research performed specifically on rice plant or rice Hbs. This is not meant as a slight against the authors, rather as an observation that current research on rice Hbs is still in its infancy and extensive literature on the topic simply does not exist. With that in mind we would suggest altering this manuscript to acknowledge this shortage of experimental evidence in the following ways.By far the majority of experimental evidence exists of Hb1, yet throughout the manuscript Hb1 is treated more-or-less equally to Hb2-Hb5. The authors should focus on Hb1 in presenting the available evidence, then in a separate section (perhaps “correlations to other Hb genes”) the authors could examine homology models and hypothetical behavior without the danger of having conjecture misinterpreted by the reader as published fact. tHb belongs to a separate class of Hbs than the other Hbs in rice (Hb1 through Hb5), and throughout the manuscript it is often treated as an afterthought. As an addendum to the above comment we would also suggest a separate section be reserved for tHb. Though little experiment information may be available for this protein, a short stand-alone section would be more informative than a series of trailing paragraphs. Great care must be taken by the authors when postulating biophysical characteristics of rice Hbs using only homology models based upon a single crystal structure. Simple variations in structure can carry significant changes in heme iron coordination, reactivity and binding affinity. If such conjecture is used in this manuscript it must be plainly stated with the caveat that this is not published fact, or experimental evidence. Additional specific comments To orient the reader it would be helpful to include a two-panel figure containing (a) the amino acid sequence alignment of Hbs 1 through 5 using Hb1 as the anchor, and (b) the alignment of tHb using Arabidopsis thaliana GLB3 as the anchor. In both panels, the differences between each Hb and Hb1 (a) or tHb and GLB3 (b) could be emphasized (e.g., with color). The secondary structure of Hb1 (a) and GLB3 (b) could be indicated as well. In the text the % sequence identity for the relevant pairs should be mentioned. In addition, are the japonica and indica sequences identical? Whether one or the other cultivar is used should be clearly indicated throughout the review (for example, in Figure 2). Apoprotein folding pathways are discussed on page 7. An additional statement as to the biological relevance of the folding pathway and information about association with the heme (when, where) would be interesting. In general, it is not possible to predict the thermodynamic or chemical properties of a heme protein based on its primary structure. The case of Parasponia andersonii and Trema tomentosa hemoglobins provides one illustration of the difficulty within the plant world. Predictions can be inaccurate even when a three-dimensional structure is available. This shortcoming of sequence analyses and modeling could be emphasized with a discussion of specific examples and used to advocate the need for additional hemoglobin research. Reaction with nitric oxide is a likely function of many hemoglobins. According to the work of Gardner and colleagues, the NO dioxygenase reaction begins with the binding of dioxygen followed by combination with NO to produce nitrate. In this mechanism, binding of NO to the iron is not necessary. In this regard, the statement on page 3 (bottom left) should be clarified. Likewise, the Hb/NO cycle as proposed by Igamberdiev and Hill involves oxyHb. In contrast, the description on page 9 suggests that one turnover occurs with oxyHb, followed by formation of Hb-NO. Speculations regarding the formation of homodimeric or heterodimeric structures should also be qualified, since the concentration of the hemoglobins (sub micromolar) appears to be much lower than the projected Kd (mM). In the \"Postulated functions” section, a proposal is made that Hb5 and tHb are O2 transporters. Could the authors elaborate on this function? (Transport to what and for what purpose, and is it consistent with the cellular concentrations?) Ligand binding is central to the function of hemoglobins. The “kinetic” section provides little such information while the second paragraph of the “Postulated functions” section offers numbers. It would be useful to consolidate the kinetic data with a table containing the measured equilibrium and rate constants for Hb1 and Hb2 (CO, NO, O2), as published in various primary references (for example, reference 97), and for Hb relatives mentioned in the text.", "responses": [ { "c_id": "1099", "date": "11 Dec 2014", "name": "Raul Arredondo-Peter", "role": "Author Response", "response": "We thank Drs. Lecomte and Johnson for evaluating the first version of this review and for providing useful comments and suggestions. We agree with them and incorporated the suggested changes into the revised version of the review." } ] } ]	1	https://f1000research.com/articles/3-253
https://f1000research.com/articles/3-72/v1	17 Mar 14	{ "type": "Research Article", "title": "Two different motor learning mechanisms contribute to learning reaching movements in a rotated visual environment", "authors": [ "Virginia Way Tong Chu", "Terence David Sanger", "Terence David Sanger" ], "abstract": "Practice of movement in virtual-reality and other artificially altered environments has been proposed as a method for rehabilitation following neurological injury and for training new skills in healthy humans. For such training to be useful, there must be transfer of learning from the artificial environment to the performance of desired skills in the natural environment. Therefore an important assumption of such methods is that practice in the altered environment engages the same learning and plasticity mechanisms that are required for skill performance in the natural environment. We test the hypothesis that transfer of learning may fail because the learning and plasticity mechanism that adapts to the altered environment is different from the learning mechanism required for improvement of motor skill. In this paper, we propose that a model that separates skill learning and environmental adaptation is necessary to explain the learning and aftereffects that are observed in virtual reality experiments. In particular, we studied the condition where practice in the altered environment should lead to correct skill performance in the original environment. Our 2-mechanism model predicts that aftereffects will still be observed when returning to the original environment, indicating a lack of skill transfer from the artificial environment to the original environment. To illustrate the model prediction, we tested 10 healthy participants on the interaction between a simple overlearned motor skill (straight hand movements to targets in different directions) and an artificially altered visuomotor environment (rotation of visual feedback of the results of movement). As predicted by the models, participants show adaptation to the altered environment and after-effects on return to the baseline environment even when practice in the altered environment should have led to correct skill performance. The presence of aftereffect under all conditions that involved changes in environment demonstrates separation of environmental adaptation and skill learning. Our results support the existence of two distinct learning modules with different adaptation properties. Therefore we suggest that adaptation to an altered environment may not be useful for training new skills.", "keywords": [ "motor learning", "adaptation", "visual rotation", "reaching" ], "content": "Introduction\n\nExperiments in haptic (Shadmehr & Mussa-Ivaldi, 1994; Gandolfo et al., 1996; Patton et al., 2001) and virtual-reality environments (Pine et al., 1996; Krakauer et al., 1999) have repeatedly shown that movement will be altered by changes in environment, and may remain altered for a short time after the original environment is restored (“motion aftereffect”). (Della-Maggiore et al., 2004; Thoroughman & Shadmehr, 2000) This observation has led researchers to suggest that either the original adaptation or motion aftereffect could be used to train skills (Rose et al., 1996; Sveistrup, 2004). Unfortunately, in most cases, the effect of the altered environment is only temporarily maintained, and thus there is no transfer of learning from the altered movement to normal skill performance (Kozak et al., 1993). We hypothesize that the reason for the lack of transfer is that task learning and environment adaptation are performed by two separate learning systems.\n\nShadmehr and colleagues (Smith et al., 2006) proposed that there are two learning systems of different time scales that underlie motor learning. These two learning systems are characterized by their time properties. The fast system responds strongly to error but also forgets rapidly, while the slow system responds weakly to error, but retains information. Recent evidence (Chen-Harris et al., 2008) suggests that the fast system has the structure of a forward internal model and the slow system could be a motor command generator. The adaptation to visuo-motor perturbations has been shown to depend on the cerebellum, and is driven by the sensory prediction error rather than the motor error (Tseng et al., 2007).\n\nWe suggest that these two systems can be separated based on their training signals rather than on their time scales. (Mazzoni & Krakauer, 2006) separated explicit cognitive strategies and implicit environmental adaptation in an experiment that tested the use of cognitive strategies to counter a visual rotation in a reaching task. Their results showed that implicit motor adaptations override explicit cognitive strategies, demonstrating the interactive nature of the two systems. In this paper, we present a mechanistic explanation through the use of a computational simulation. In particular, we suggest a system that responds to sensory prediction error and learns the structure of the sensory-motor dynamic environment (“fast system”), while another system responds to task performance error and learns the elements needed to perform a task (“slow system”). We further suggest that the two systems have very different generalization properties, so that while the sensory prediction error system can generalize broadly across the environment, the performance error system does not generalize to dissimilar tasks. These properties are consistent with two different and simultaneously-active learning systems, and we will simulate a simple model of this structure to compare with human data. The proposed structure is similar in spirit to a model originally proposed by Doya, in which there are separate neuroanatomical regions for motor planning and for adaptation to changes in dynamics (Doya et al., 2001).\n\nMany experiments that use altered visuo-motor environments confound the two types of error, so that performance error is caused by sensory prediction error. In such cases it is not possible to distinguish the two learning systems. In order to distinguish the two systems we need to test the effect of sensory prediction error when performance error is zero, and the effect of performance error when sensory prediction error is zero. By doing so, we will show that the two systems have very different generalization properties, and therefore cannot be implemented by the same network.\n\nWe use a very simple experimental paradigm. The “skill” that we test is the ability to make straight reaching movements to different targets on a pen tablet. This is a very simple and overlearned skill, but it provides a sufficient model for testing the hypothesis. Here we consider movements to different targets to represent different skills, since different movement directions require significant changes in the pattern and timing of muscles used. The “environment” that we test is the relation between hand movement on the pen tablet and the visual image of movement that is seen by the subject. Different rotations of the displayed hand movement with respect to the true hand movement are considered to be different sensory-motor environments.\n\nWe compare the results to three simple model structures for skill learning and environment adaptation (Figure 1). Structure 1 consists of a single network, and structures 2 and 3 have increasingly more complex structure. Each has different generalization properties. Structure 1: Skill learning and environment adaptation are performed by a single shared network for all tasks (directions of hand movement) and all environments (visual rotations). This structure predicts that adaptation to a new environment will change performance on multiple targets. It also predicts that practice on one target will affect performance on other targets even without a change in environment. Thus both the environment and the task will generalize across multiple targets, and environment learning will have a broad effect on task learning. Structure 2: Task learning and environment adaptation are performed by a single distinct network for each target. This structure predicts that adaptation to a new environment will change performance only on the particular target practiced in that environment. Thus neither the environment nor the task will generalize across multiple targets, but environment learning will have a focused effect on task learning. Structure 3: Task learning is performed by a separate network for each target, but environment adaptation is performed by a single shared network. This structure predicts that adaptation to a new environment will change performance on multiple targets, but practice on one target will not affect performance of other targets. Thus the environment will generalize across multiple targets, but the task will not, and environment learning will have no effect on task learning because it is performed by a completely different subsystem. Note that we do not test the fourth implied possibility, in which the task generalizes across multiple targets but the environment does not, because this is not consistent with known previous results (Goodbody & Wolpert, 1998). Only structures 1 and 2 could permit environment adaptation to be useful for training tasks, since only in these structures are the parameters modified by environment adaptation also used for tasks (see Figure 1).\n\nA. Structure (1) is a single learning network that adapts to both changes in skill and environment. The same system learns based on errors in the observed task. B. Structure (2) is a motor program model. Learning is performed by separate network for each task based again only on observed error. C. Structure (3) is a two system-learning model. This model splits into two groups of systems, where there is a separate system for each task, learning based on observed error, but a single inverse model for control of the environment, learning based on prediction errors.\n\nIn this study, we will reject structure 1 by showing that learning one task does not lead to aftereffects on a second task, and therefore show that sufficiently different tasks do not share parameters and are probably learned by distinct networks. Furthermore, we will reject structure 2 by showing that learning a new environment does lead to aftereffects when the environment returns to baseline, and therefore that multiple environments are learned by a single network with a single set of parameters. Our results support structure 3 by showing that learning one environment leads to aftereffects in a different environment, but learning one task does not affect learning of another task. Since adaptation to sensory-motor error generalizes broadly while task learning generalizes narrowly, we claim that environment learning and task learning cannot be implemented by the same network. An interesting consequence of the independence of the two systems is that when the task error is zero but the sensory-motor mismatch is nonzero, adaptation reduces the mismatch even at the expense of worsening the task error, confirming the results of (Mazzoni & Krakauer, 2006). Therefore adaptation to the environment is controlled independently of the task error, and our results will support the existence of two different learning systems that respond to two different types of error.\n\nMotion aftereffect paradigms have provided useful results concerning generalization of adaptation to different task or environment parameters. (Krakauer et al., 1999; Shadmehr & Moussavi, 2000; Vetter et al., 1999; Krakauer et al., 2006; Hwang et al., 2006) Here, we show that the type of generalization depends on the type of error that drives learning, consistent with the hypothesis of two different learning systems that are distinguished by the error to which they respond and the way in which they generalize across the environment.\n\n\nSimulations\n\nOne example of each of the three models was simulated. To do this, several assumptions were made. We assumed that the participants had already learnt the dynamics of their arm and know the motor commands for reaching movements. Therefore, the learning of the arm dynamics was not included in the model. Furthermore, we assumed that the participants would have already learnt what trajectory would solve the problem optimally from previous experience. Based on physiological studies, human movements are observed to have a bell-shaped velocity. (Gordon et al., 1991; Bizzi & Abend, 1983) This type of velocity profile has been shown to be optimal for many cost criteria such as the minimum-jerk criterion in optimal controlled reaching (Nagasaki, 1989). For simplicity, the optimal controller (trajectory generator) was modeled with a desired trajectory generator, which generates a straight-line trajectory to the target with a bell-shaped velocity profile. The bell-shaped profile used is a normalized truncated Gaussian distribution function, which is lowered so that it has zero initial velocity.\n\nThere were three components used in the models: the trajectory generator, the environment adapter and the online feedback controller. The use of these elements is illustrated in Figure 2. From Figure 2 we see that in models A and B there is one environment adaptation module for every trajectory generation module. Therefore, in the simulations of models A and B we used the same network for trajectory generation and adaptation to the environment. Model A has a single network for trajectory generation and environment adaptation, while model B has a separate network for each task that learns both the trajectory and the environment. The separation of learning networks for each task is a simplification in modeling to replicate a task generator that generates a command trajectory for each desired target in a continuous fashion. With this simplification, we could use basis function networks to model task learning rather than other more complicated networks. In model C, there is a separate trajectory generator for each task but only a single environment adaptation module, so two different types of network must be used. In the trajectory generator module, the error signal is based on performance error, the error observed by the participant during each trial. For the environment adapter, the error that trains the network is based on the prediction error, the difference between the participant’s anticipation of movement and the actual observed movement.\n\nP (Plant) is the matrix that represents the rotation of the visual feedback. P-1 is the internal inverse model of the plant. The desired trajectory generator uses a bell-shaped velocity profile to generate the desired trajectory. The “Time diff” block calculates the velocity sequence that would have produced the planned trajectory using the planned position sequence. Using the planned velocity sequence as the input command, the online correction takes the delayed error as a proportional correction term to give the new velocity to guide where the cursor should go relative to the current location. The learning networks in the models are basis function (BF) networks where each basis function is an increasing order monomial of time. A. Detailed simulation model for model A (simple learning model). B. Detailed structure for model B (motor program). C. Detailed structure for model C (two-system learning model).\n\nFor any task k, the desired trajectory generator creates a desired trajectory x[k]d(t) and y[k]d(t). This trajectory is compared with the observed feedback to generate the error signals xe(t) and ye(t). At each trial, the learning network produces the planned trajectory, xp(t) and yp(t). xp and yp are the input to a feedback controller. The feedback controller combines the planned trajectory and the error xe(t) and ye(t) at each point in movement in order to generate a motor command xc(t) and yc(t). The plant takes the motor command xc(t) and yc(t) (which is just a desired position on the pen tablet) and transforms it into the observed position xs(t) and ys(t) that is then available as sensory (visual) information. The plant transforms the motor command into the sensory output by rotating, so that [xsys]=P(θ)[xcyc], where P(θ) is a 2×2 rotation matrix.\n\n\n\nThe sensory output is compared with the desired trajectory to generate the error signal xe = xd - xs, ye = yd - ys. The error signal is used in real-time for feedback control, and it is used at the end of each movement attempt to update the trajectory generator module. Assuming that the desired trajectory (xd, yd) is known, the goal of the trajectory generator is to minimize the cost function, ε, the norm of the error signal integrated over time.\n\n\n\nIn addition to the above elements, model C includes a separate environment adaptation module that modifies the motor command xc yc and changes it to x′ y′c in order to compensate for changes in the plant (rotation). It performs this modification by using a plant inverse P–1(θ) that predicts the correct motor command x′c y′c for any desired sensory output xs, ys. The plant inverse is learned by approximating the plant “forward model” from x′c y′c to xs, ys and then inverting the resulting 2×2 matrix. Note that in models A and B, a change in the environment results in a change in the trajectory generator because of the increased performance error xe and ye. In model C, a change in the environment will be compensated by the environment adaptation module and thus the trajectory generator module will not change.\n\nThe input to the trajectory generator was the x and y coordinates of the target location (xt, yt) as shown on the display. Using the given target (xt,yt), the desired trajectory generator generated a trajectory (xd(t), yd(t)) that it “hopes” to see on screen, based on the assumptions mentioned above (straight and bell shaped velocity).\n\nThe learning network was programmed as a basis function neural network. The basis functions used were polynomials of time up to degree n. Let Wx ∈ ℜn and Wy ∈ ℜn be unknown weight vectors. Then the output of the trajectory generator was written as\n\nxp(t) = Σi Wxi × Φi(t) and yp(t) = Σi Wyi × Φi(t)\n\nwhere Wxi and Wyi were the ith elements of the Wx and Wy vectors and Φi(t) is the ith degree monomial of t (Φi(t) = ti). The outputs xp(t) and yp(t) were then passed to the rest of the learning model. The outputs xp(t) and yp(t) specify the trajectory input provided to the feedback controller.\n\nThe weight vectors Wx and Wy were trained using the errors (xe(t), ye(t)) between the desired trajectory (xd(t), yd(t)) and the trajectory of the movement observed on the screen (xs(t), ys(t)). The training algorithm is the Widrow-Hoff “least mean squares” (LMS) training algorithm that is known to converge for stationary inputs. (Widrow & Hoff, 1960; Widrow et al., 1976) Unfortunately, when used in a control system, the inputs are non-stationary and thus convergence of LMS is not guaranteed. Nevertheless, this is a commonly used algorithm that has been shown to perform well in adaptive control tasks (Sanger, 1991; Sanger, 1994; Sanner & Slotine, 1992) and it provides one of the simplest models of motor learning (Schweighofer & Arbib, 1998; Berthier et al., 1993).\n\nxe(t) = xd(t) – xs(t) and ye(t) = yd(t) – ys(t)\n\n∆Wxi = λ Σj xe(tj)Φi(tj) and ∆Wyi = λ Σj ye(tj)Φi(tj) for all i\n\nWx = Wx + ∆Wx and Wy = Wy + ∆Wy\n\nλ was the learning rate of this system. The weights were updated after each trial, based on the errors from the whole movement. In models A and B, the output xp and yp represents both task learning and adaptation to the environment, since a change in the environment or the desired trajectory will lead to a change in error xe and ye that will modify the weights in the network. In model C, there is a separate stage of environment adaptation and thus this initial network is responsible only for adapting to changes in the task. In models B and C, the multiple neural network structure was simulated by storing multiple weight vectors W(k)x and W(k)y which can be trained or retrieved when needed for any particular task k.\n\nThe environment adapter built an internal model of the environment, Pˆ, giving predictions, xˆs, yˆs. The internal model was inverted to provide the environment inverse Pˆ–1. This gave the learning system a way to anticipate the rotational field and attempted to “undo” its effect. At each trial, the input to the environment inverse was the output from the feedback controller (xc(t), yc(t)). The model inverse produced the modified command trajectory (x′c(t), y′c(t)) that the system anticipated could invert the plant. This signal is then fed through both the plant and the plant model. By computing the error between the plant output and the plant model output (ep), the plant model Pˆ, could be trained. Training of the plant model was done through the LMS algorithm. Since the posed problem was essentially solving a linear regression, the LMS is guaranteed to converge, assuming that the learning rate is not too large.\n\n\n\nwhere αe was the learning rate of the system.\n\nAs a human participant would, the system in the simulation should also be allowed to correct errors online. Therefore, an online feedback correction controller was implemented. The planned trajectory (xp, yp) from the trajectory planner was used as input to the controller and it was also given a feedback of the error “observed” on screen (xs, ys). This error was calculated based on the desired trajectory (xd, yd). The online feedback is delayed by 100ms, the same order of magnitude of recorded human visual reaction time. (Fischer & Ramsperger, 1984) The controller output was proportional control using the delayed feedback with a feedback gain γ, added to the feedforward control generated using xp and yp.\n\nxc(t) = xc(t-1) + [xp(t) – xp(t-1)] + γxe(t-td)\n\nyc(t) = yc(t-1) + [yp(t) – yp(t-1) ] + γye(t-td)\n\n\nExperimental methods\n\nIn a visual rotation (VR) setting, there are two aspects to any task: vision (what the participant sees) and motor (the participant’s actual movement). In our experiment, the participants observed the visual feedback of their movement on a LCD monitor and performed the movement using their unseen hand under the monitor. We manipulated the relationship between movement and visual feedback in order to force the participants to adapt to a new motor-sensory map (environment). We determined whether this adaptation interfered with performance of a previously-learned task (straight line movements in different directions). In one experimental manipulation, participants were asked to make a movement that appeared visually the same, for example, moving toward the same target on the screen, but required a different hand movement due to a change in the visual-motor map. We will refer to this as “same task (target) different environment (visuo-motor map)”. In a second experimental manipulation, we asked the participant to make a movement to a different target but without a change in the visual-motor map. This required a change in the actual (unseen) hand movement, so we refer to this as “different task same environment”. In a third experimental manipulation, we asked the participant to make a movement to a different target, but the visual-motor map was changed so that successful performance occurred for the same hand movement in both cases. In other words, the change in target and the change in visual-motor map were in exactly opposite directions and cancel each other out. We refer to this case as “different task different environment”, although the required hand movement did not change. The movements were recorded using a pen tablet (Wacom, Intuos 2 XD-0912-R, Saitama, Japan) connected to a personal computer (Fujitsu, Lifebook T4010, Tokyo, Japan). The participants were asked to complete four short experiments. In each experiment, there were three blocks of 20 reaching trials, reaching from the center of the screen to a target location on a circle. The first and third block were always under the same experimental condition and in the second block, we changed the vision and/or the motor aspect of the task. The experiment was designed to determine whether the condition in the second block interfered with performance of the skill attempted in the first block by causing aftereffects at the beginning of the third block. Thus the three blocks are namely, baseline, interference, and re-adaptation. The outcome measure was a comparison between baseline (before the interfering condition) and re-adaptation (after the interfering condition). The four experiments were carried out in a pseudorandom order for each subject, such that each subject completed all 4 experiments in a randomized order.\n\nSince straight-line reaching is a heavily practiced skill for most subjects, the visual environment was rotated 10 degrees clockwise in the baseline condition. The baseline condition was therefore no more familiar to subjects than the interference conditions, so subjects were unable to use their extensive prior experience with reaching to override errors induced by the adaptation. Changes in the rotation feedback (environment) were made relative to the baseline environment.\n\nIn experiment 1 (same task, different environment), the feedback of the movement in the interference block was rotated counterclockwise by 20° relative to baseline (see Figure 3). The target location on the visual display was not changed. This was a typical aftereffects paradigm, where the subject was asked to perform the task with the same specification, but with a change in environment. Two things changed in the experimental condition between the first and second block of the experiment: the visuo-motor map and the movement the subject was required to make. In order to tease apart which is the main cause of the aftereffects, we designed experiment 2 and 3 to test each aspect. In experiment 2 (different task, same environment), the visual feedback was not rotated, but the target location moved in the interference block. This experiment was designed to look at interference between learning different hand movements. In experiment 3 (different task different environment), the feedback of the movement in the interference block was rotated counterclockwise by 20° relative to baseline. The target location (task) in state B was also rotated by the same amount in the opposite direction to keep the required arm movement the same. This experiment was designed to look at the effect of changes in the visuo-motor map (environment) without a change in the hand movement required to solve the problem. However, in order to test this we had to change the visual display. Therefore, a fourth experiment, Experiment 4 (different task, different environment) was designed as a control for experiment 3 where the visual display was not changed throughout the three blocks. The purpose of this control is to ensure that the effects in experiment 3 were not simply due to the altered sensory display. In the baseline condition, instead of asking the subjects to reach to the target location, we placed the target at 20°, and asked the participant to reach to 40°. Additional feedback was given in form of a score. The score was calculated as 100 - the target error (in degrees). The target error was calculated at the point when the subject’s hand crossed the circle where the target lies. In the interference block, the target was still kept at 20° and the feedback of the movement was rotated counterclockwise 20°. The participants were asked to reach to the target to keep the movement the same. Therefore, the visual display did not change although the target and environment did. We used this experiment to verify whether the results observed in experiment 3 could be explained by changes in the visual display. Through these experiments, we can separate the effect of target (the intended movement) from sensory stimulus (what appears on the screen).\n\nIn each of the four experiments, there were three blocks, with 20 trials each. In the figure, the monitor represents what the participants saw during the experiment. The rectangle below represents the subjects’ arm movement. The dark circular dot represents the starting location, and the light colored dot represents the target presented to the participants during the experiment. The line in between the dots indicates the desired movement as observed on screen. Two striped marks on the side of the screen always indicate the “west” direction on the tablet, which was used as an indicator to the participants to let them know which environment they are in. Figures A, B, C and D show the experimental setup for experiment 1, 2, 3 and 4 respectively. E and F show details of each experimental block.\n\nBecause subjects may make large errors following an unexpected change in the visual rotation, they were provided with a visual indication of the environment, and this indication also served as a warning when a change occurred. The indicator on screen marked the “west” direction on the pen tablet. Therefore, when the visual feedback was rotated, the indicator moved on the screen according to the rotation field. The participants were instructed to reach out from the center as fast and as straight as possible. If they completed the movement and reached the target within a time limit (1 s), the target would flash orange to indicate a success. After each trial, the participant was guided to move the cursor back to the starting location without direct feedback of the cursor location. On the screen, the participants were shown a circle whose radius represents the distance between the subject’s cursor and the starting location. The participants were told to move their cursor to minimize the size of the circle in order to move the hidden cursor back to the starting point.\n\nTen participants with no history of neurological diseases were recruited from Stanford University. The participants were between the age of 23 and 27, six males and four females. All participants were right-handed and performed the experiments with their right hand. Follow-up tests with another group of participants were carried out after the conclusion of the first study. The follow-up study was on the same rotation paradigm only with larger changes in rotation angles. For the follow up study, eight adult participants (average age: 25.4, five females and three males) were recruited for further tests. These eight participants did not participate as a subject in the first group. All except one of the follow-up study participants were right handed. All participants performed the tasks with their dominant hand. All procedures were approved by the Stanford University Institutional Review Board. Participants signed written consent for the experiment and HIPAA authorization for the use of personal data.\n\nData analysis was performed in MATLAB. The primary data were the samples of the pen tablet position sampled at 50Hz. The initial direction of movement was calculated as the angle of the line connecting the start location to the point of maximum velocity (Krakauer et al., 1999). For each trial, the average and standard deviation of the initial reach angle for all participants were calculated. The extent of an aftereffect in each experiment was determined by comparing the first trial in state C with the baseline statistics computed from the last ten trials of state A. The baseline statistics, means (μ) and standard deviations (σ), were calculated for each experiment per participant. Using the measurement from the first trial of state C (xc1), the magnitude of the aftereffect was calculated as a z-score.\n\n\n\nA larger z value is associated with a greater aftereffect. The presence of an aftereffect was tested statistically by performing a hypothesis test with α = 0.05. When z > 1.96, the null hypothesis (xc1 belongs to the baseline distribution) was rejected, and we asserted the presence of a statistically significant aftereffect.\n\n\nResults\n\nBefore the simulation of the experiment, the model was allowed to “practice” straight lines from the center to the various target location, without the rotation of the “visual feedback” (not shown in the figure). The models practiced several hundred straight lines movements to targets at 0° (straight up), 20°, 40° and 60°. This allowed the model to have relatively similar experience as a human subject, where humans are assumed to already know how to make straight lines to the various targets in the normal environment. The learning rates in the models were tuned by starting with very small values, and increasing them until the system was able to learn in approximately the same speed as the human subjects (within 20 trials). The online feedback gain was tuned to adjust the trajectories such that the trajectories would end at the target location. The system was approximately overdamped even if there was large initial error.\n\nThe simulations showed a small learning curve at the beginning of experiment 1. This was because the model has to learn to adapt to the +10° rotational field. The simulation started with doing experiment 1 and we did not program a break in between the experiments, so the computer could retain the +10° field learnt from before and did not show a learning curve in the beginning of experiment 2 and 3.\n\nAccording to the calculations outlined earlier (results shown in Table 1, raw data in dataset 1), in experiments 1 and 3, ten participants showed significant aftereffects that reached statistical significance (≥ 0.05); in experiment 4, nine participants showed aftereffects. In experiment 2, only three participants showed a significant aftereffect. In experiment 1, all subjects showed transient aftereffects both at the onset of the altered environment (first few trials of state B) and at the return to the baseline environment (first few trials of state C). This agrees with previous results and shows that subjects adapted to the altered environment in a way that suggests the presence of an adaptive internal model (Wolpert et al., 2001; Kawato, 1999). Aftereffects were also seen in experiment 3 and 4. Note however, that although subjects achieved the desired performance in the altered environment (at the end of state B), the return to the baseline environment caused worsening of performance (beginning of state C) that only gradually returned to its original baseline. Aftereffects were not observed in experiment 2.\n\nRepresented in the table are the aftereffect z scores calculated for each participant for the 4 experiments (Exp). The bolded numbers are the ones considered significant under the assumption of α=0.05 (z > 1.96). The significant column (Sig.) in the table represents the number of z scores (out of 10) that are significant in that experiment. the table also includes results from the three models: + indicates a presence of aftereffects prediction and – indicates an absence of aftereffects prediction.\n\nBy comparing Figure 4 and Figure 5, we see that models A and B do not match the experimental results. Only model C is consistent with human results in all of the experiments and predicts aftereffects in experiments 1, 3, and 4, but not 2. Figure 6 shows several typical trajectories from a participant and simulations of model C. The terminal “hook” is due (in the model) to the feedback controlling online correction.\n\nResults plotted are the initial angle (in degrees) of the simulated movement observed on screen against the trial number in that experiment. The solid black line represents the computer simulation results; whereas the dotted grey line represents the target presented for that trial. Experiment 4 was not simulated due to the nature of the experiment being very similar to experiment 3. To a computer simulation, experiment 3 and experiment 4 are the same as the difference between the two experiments comes from difference in visual display. The first column (A, D, G) are the results from model A, second column (B, E, H) are results from model B and the third column (C, F, I) are from model C. The first row (A, B, C) are results for experiment 1, the second row (D, E, F) are results for experiment 2, and the third row (G, H, I) are results for experiment 3.\n\nThe initial direction measurement of the movement observed on screen. This is the trial by trial average of all ten subjects. The error bars mark the standard deviation amongst the subjects and the dotted grey line represents the desired target angle observed on screen. The vertical axis is the initial direction measured in degrees and the horizontal axis is the trial number. A. Experiment1, same task different environment. B. Experiment 2, different task same environment. C. Experiment 3, different task different environment. D. Experiment 4, different task different environment (same display target).\n\nThe trajectories are taken from Experiment 3. The participants plot contains the trajectories from all ten participants for that particular trial. A, C, and E are from participants. B, D, and F are from simulations. A and B are one of the baseline trajectories. C and D are from the interference state. E and F are from the readaptation state. In figures C–F, the black lines are from the beginning of the state, and the grey lines are from the end of the state.\n\nIn the follow-up study, a second group of participants (8 in total) were recruited to study the generalization of aftereffects in larger rotation angles. We repeated our first experiment with a 90° rotation between environments. The targets were also further apart. The targets were located at 90°, 180° and 270° rather than 20°, 40°, and 60° respectively. We call this the 90 degrees experiment. The results in the 90 degrees experiment are presented in Figure 7 and the statistics in Table 2, with the raw data in dataset 2.\n\nThe figure shows initial direction measurement of the movement observed on screen. This is the trial by trial average of all eight subjects. The error bars mark the standard deviation amongst the subjects and the dotted grey line represents the desired target angle observed on screen. The vertical axis is the initial direction measured in degrees and the horizontal axis is the trial number. A. Experiment 1, same task different environment. B. Experiment 2, different task same environment. C. Experiment 3, different task different environment. D. Experiment 4, different task different environment (same display target)\n\nRepresented in the table are the aftereffect z scores calculated for each participant for the 4 experiments (Exp). the bolded numbers are the ones considered significant under the assumption of α=0.05 (z > 1.96). the significant column (Sig.) in the table represents the number of z scores (out of 8) that are significant in that experiment.\n\nThe results in the 90 degrees experiment were not as strong when compared to the original 20 degrees experiment. However, the conclusion still stands that in experiment 1, 3, and 4, the participants showed stronger aftereffects than in experiment 2. The initial errors that occurred in the interference state of experiment 2 showed the environment learner’s limited ability to generalize to targets that are far away from the initial training. Yet the observation that training of a new target in the second environment did not interfere with the performance of the initial target upon return to the re-adaptation state reinforces the separation of task learning and environment adaptation. Aftereffects were observed in experiment 3 and 4 indicating that the training in a different environment at a target 90o away from the initial target interfered with the initial training.\n\n\nDiscussion\n\nConsistent with prior force and rotation field studies (Martin et al., 1996; Shadmehr & Mussa-Ivaldi, 1994; Gandolfo et al., 1996; Patton et al., 2001), participants showed transient aftereffects when they returned to the original experimental condition after practicing in a different rotational field (experiment 1). However, aftereffects did not occur following a change in the task (experiment 2). Aftereffects did occur in a different rotational field even when the task was adjusted so that the required hand movement did not change between conditions (experiment 3). Aftereffects also occurred in a different rotational field when both the visual display and the required hand movement did not change (experiment 4).\n\nAftereffects can be considered a type of interference between conditions in which the prior condition affects the initial performance in the subsequent condition. Our results show that interference between conditions occurs if and only if there is a change in the rotational field (the environment). A change in the target task is not sufficient, by itself, to cause interference or aftereffects. Since changes to the environment interfere with each other (experiment 1, 3, and 4), the results suggest that there is only a single environment internal model that adapts and re-adapts (thereby showing aftereffects); whereas there are multiple independent modules for task performance (experiment 2).\n\nThe most important observation comes from experiment 3, in which environment adaptation exactly compensated for errors in task performance. In this experiment, adaptation to the rotation of the visual environment caused the hand movement to solve the desired task. Immediately after the visual environment was returned to the baseline, the next attempted hand movement should have low error. If environment adaptation and task learning shared a common mechanism, then the low task error should have resulted in continued good performance. However, as soon as the environment returned to baseline, there was a higher than expected sensory-motor mismatch, and the subjects responded by adapting to the mismatch, even though this resulted in worsening task error. Therefore environment adaptation is not controlled by task performance error. This result is similar to the results of Mazzoni and Krakauer and probably is due to the same mechanism (Mazzoni & Krakauer, 2006). Our results strongly imply that the two systems use different learning mechanisms. An important consequence is that for this very simple set of tasks, adaptation to an altered environment is not useful for training task performance.\n\nThis conclusion is supported by the model simulations. From Figure 4 and Figure 5 we see that model A is not consistent with the human data since there are aftereffects in experiment 2 in the model but not in the human data. Model B is not consistent since there are no aftereffects in experiment 3 in the model but there are in the human data. Only model C correctly predicts the presence of aftereffects in experiments 1 and 3 but not in experiment 2. Model C includes separate networks for each task, but a shared network that adapts to the environment. Thus the match between data and simulations of model C supports our hypothesis.\n\nThese experimental results are consistent with prior results on generalization (Vetter et al., 1999; Krakauer et al., 2000). Previous studies concluded that environment adaptation to visuomotor rotation has good generalization properties for targets within 45°. This is consistent with the aftereffects the participants and the model simulations showed in our 20° rotation experiments. However, based on these previous results, aftereffects were not consistently observed for the targets were more than 45° apart from each other. In order to test the generalization of the model to targets more than 45° apart, we performed the 90 degree experiment. If there were no generalization of the environment adaptation to targets more than 45° apart, there should be no aftereffects in experiments 3 or 4, where the participants were trained in a different environment at a target that was 90° away from the target used in the first environment. Since aftereffects were observed in the human subject results in experiments 3 and 4, we infer that environmental adaptation can be generalized to targets larger than 45° apart.\n\nAn important consequence of these experiments is that training in the rotated environment may not be helpful for improving task performance, since the rotated environment leads to modification of the environment adaptation module but not the task generation module. This is directly seen in the human data for experiment 3, in which the motor task remained the same under all conditions. Performance transiently worsened when the rotation returned to baseline, even though no change in hand movement was required to achieve correct performance. In the simulation model, this occurs because there are two different types of error that are used to train the two learning modules. When the baseline rotation is restored in experiment 3, the performance error ex, ey is zero, since the initial hand movement is correct. However, the plant inverse error, ep, is nonzero and thus the plant inverse learns (and motor performance changes) even though there was no performance error. This is an important distinction between the two systems. Task learning is driven by performance error, while environment adaptation is driven by predictions of the environment response, independent of the desired task. Therefore this model suggests that virtual reality adaptation may be insufficient to train task performance.\n\nOur results are consistent with the common observation that a skill can often be performed in a different environment with substantially less retraining than originally required to learn the skill, described by Krakauer and colleagues as task-specific savings (Krakauer et al., 2005). Our results are also consistent with the observation of transient aftereffects after changing the mechanics of the environment. Our results are also consistent with the ability to learn multiple new skills without forgetting previously-learned skills. Although previous models have addressed skill learning or environment adaptation separately, our results and model simulations represent one of the first quantitative studies to examine their interaction. A recent study (Mazzoni & Krakauer, 2006) on rotational field experiments concluded that implicit adaptation to a visuomotor rotation overrides the explicit strategies given by the experimenters. Despite the use of explicit cognitive strategies that opposes the visual rotation, experiment participants unconsciously adapted to the rotational field, making increasing errors to the target. The rate of adaptation was the similar with and without the explicit cognitive strategies, showing that implicit adaptation occur independent of the use of explicit strategies. The “implicit adaptation” is equivalent to the environment learning network in our model, and “explicit strategies” are equivalent to task learning in our model. Their results are consistent with our model, showing in fact two types of learning interact in the learning of visuo-motor adaptations. Our model provides a good framework to capture the two types of contribution to motor learning.\n\nOur two systems model was also consistent with the fast and slow adapting systems as Shadmehr and colleagues proposed (Smith et al., 2006). The two-rate learning model has been used to explain task interference (Sing & Smith, 2010), generalization (Tanaka et al., 2012), savings (Zarahn et al., 2008) and retention (Joiner & Smith, 2008). We believe that our two system model will offer another perspective in differentiating the two learning systems. Our environment adaptor would behave similar to the fast adapting system, and the trajectory generator would behave like the slow adapting system.\n\nThere are several weaknesses of the current model that need to be addressed in future experiments. The model does not explain the observation that learning two very similar skills can generate interference. Such an observation could be incorporated in a model in which different tasks are represented not by a set of discrete motor programs but by a parameterized or “fuzzy” mixture of motor programs perhaps using a local basis function network (Poggio & Girosi, 1990). The model also does not explain the observation that after extensive practice it is possible to switch between two environments (e.g. prism glasses) almost instantly (Martin et al., 1996; Shadmehr & Wise, 2005). This observation could be incorporated using an environment learning model that can learn to respond to cues indicating a change in the environment. Our model does not yet explain differences in performance following blocked or interleaved practice (Simon & Bjork, 2001). This would depend upon the details of the task learning and environment adaptation algorithms. For instance, in certain neural network algorithms blocked practice (as in our experiment) might be more likely to retrain existing weights to fit the most recent condition, while interleaved practice might be more likely to fit the network output so that it performs correctly in multiple different conditions.\n\nThe two learning systems proposed in the model are analogous to two types of control systems. The task learning system can be compared to an optimal controller that learns a desired trajectory that will achieve the task goal. The environment learning system can be compared to an adaptive controller that learns the motor commands required to achieve the desired trajectory in the current environment. This is similar to the differing neuro-anatomical modules in Doya’s proposed framework (Doya et al., 2001). In this context, it is interesting to speculate whether errors in the adaptive controller could facilitate or interfere with learning in the optimal controller. If so, then it might be possible to use our model to redesign current virtual-reality training programs so that a change in the environment that leads to a change in the adaptive controller might also facilitate task learning. In future studies we plan to study this potential interaction, and we plan to investigate the interaction of the two learning systems for more complex tasks in which the dynamics of movement must be learned.\n\n\nInformed consent\n\nParticipants signed written consent for the experiment and HIPAA authorization for the use of personal data.\n\n\nData availability\n\nfigshare: Data from motor learning experiments, doi: 10.6084/m9.figshare.957526 (Chu & Sanger 2014)", "appendix": "Author contributions\n\n\n\nDr. Chu contributed to the conception, design, acquisition, analysis and interpretation of data; drafting and revising of the article; and final approval of the manuscript. Dr. Sanger contributed to the conception, design, and interpretation of data; drafting and revising of the article; and final approval of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was conducted as part of a graduate research work at Stanford University, supported by grant 1103725-100-PAAWK from the National Institute of Health to Dr. Sanger, as well as by the Stanford University BIOX bioengineering graduate student fellowship. We acknowledge additional funding from the Crowley-Carter Foundation and the Don and Linda Carter Foundation.\n\n\nAcknowledgements\n\nThe authors would like to thank Dr. Denise Y. P. Henriques for her invaluable comments and discussion.\n\n\nReferences\n\nBerthier NE, Singh SP, Barto AG, et al.: Distributed representation of limb motor programs in arrays of adjustable pattern generators. J Cogn Neurosci. 1993; 5(1): 56–78. 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PubMed Abstract \| Publisher Full Text\n\nVetter P, Goodbody SJ, Wolpert DM: Evidence for an eye-centered spherical representation of the visuomotor map. J Neurophysiol. 1999; 81(2): 935–939. PubMed Abstract\n\nWidrow B, Hoff ME Jr: Adaptive switching circuits. IRE Western Electric Show and Convention Record. 1960; 4: 96–104. Reference Source\n\nWidrow B, McCool JM, Larimore MG, et al.: Stationary and nonstationary learning characteristics of the LMS adaptive filter. Proc IEEE. 1976; 64(8): 1151–1162. Publisher Full Text\n\nWolpert DM, Ghahramani Z, Flanagan JR: Perspectives and problems in motor learning. Trends Cogn Sci. 2001; 5(11): 487–494. PubMed Abstract \| Publisher Full Text\n\nZanone PG, Kelso JAS: Evolution of behavioral attractors with learning: nonequilibrium phase transitions. J Exp Psychol Hum Percept Perform. 1992; 18(2): 403–421. PubMed Abstract \| Publisher Full Text\n\nZarahn E, Weston GD, Liang J, et al.: Explaining savings for visuomotor adaptation: linear time-invariant state-space models are not sufficient. J Neurophysiol. 2008; 100(5): 2537–2548. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "5345", "date": "04 Jul 2014", "name": "Gelsy Torres-Oviedo", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title is appropriate for the work. The abstract needs to be revised to improve its clarity. Specifically, the authors need 1) to include a brief definition for skill learning and 2) to clarify that after-effects equate to poor skill performance in their study since literature presents some other cases of after-effects leading to improved movements.Overall the article content is clearly explained and very well written. There are only a few general conceptual points that should be addressed. Authors should consider revising their conclusions based on these points.It is unclear how different the reaching directions need to be, for them to be considered the same tasks or skills? A common patients' deficit is their perseverant behavior from one situation to another (even when this is inappropriate). Literature shows this motor perseverance appears beneficial when experiencing environmental adaptations. Given that patients continue doing the adapted motion when the external perturbation is removed, it ultimately leads to long-term changes in their movements with repeated exposure. The authors are very adamant in their conclusions that environmental adaptation cannot be used as a rehabilitation intervention, yet their two-mechanism model does not seem to include this perseverance feature of patients' behavior. Kindly elaborate on this. In their theoretical framework, authors present a single performance error and they do not seem to differentiate between self-generated and externally generated performance errors. Possibly this simplification limits their model to predicting behaviors during visual tasks but it would fail at explaining behaviors in proprioception-driven tasks, like reaching in a force field or walking on a split-belt treadmill.Other comments in order of appearance:Page 4: Link description of model predictions to their visual representation on Fig 4. Conceptual question in Figure 2: It is not clear why the desired trajectory generator is not part of the controller block in which all the trajectories are generated. What are the learning network blocks computing? Illustration points in Figure 2: The \"environment adaptation module\" in Model C could be better indicated, since it is not very clear that it is missing in Model A and B. The equations for the online correction on Fig. 2 do not match the ones on the text (page 7). Kindly add V and V' on the text or change equations on Fig. 2. Page 6: xc' and yc' are missing the c. Page 6, under Trajectory generator section: It is not well explained what j and i are on the equations. Page 6 under environment adapter section: Authors should consider adding the equation in which xc' and yc' = Phat-1 [xc yc] and the learning rule for P, as they do for indicating the update of W. Page 7: Authors randomize the order of the 4 experiments to account for the order of the task. However, it is not very clear if repeated exposures of the adaptation task changes subjects behaviour. Page 7, under procedure: In the explanation of experiment 4 it is unclear if subjects were able to see the cursor or not. Page 7: Authors equate the intended movement to the target position. However, the intended movement could be different from the target position when subjects are told to aim at a different direction (as done by Mazzoni and Krakauer, 2006). Please clarify. Page 9, under data analysis section: Authors perform z-score to determine if after-effects are statistically different from baseline. It is unclear whether similar results are obtained if means (baseline vs after-effect) are compared.", "responses": [ { "c_id": "1106", "date": "10 Dec 2014", "name": "Virginia Chu", "role": "Author Response", "response": "Thank you for your valuable comments and we have addressed them in our new revision in detail, listed below.Thank you for your suggestion on the abstract. It has been edited accordingly. You have raised a very interesting topic on the delineation between same and different tasks/skills. As there is no consensus in the scientific community regarding the criteria for defining tasks that are the same or different, this provides for an interested discussion. We have added a discussion on this topic We agree with your assessment of patient deficits. Our simulation focused on understanding typical adult motor performance, and thus did not address motor performance in motor disorders. We believe that the type of perseverant behavior in patients can be also understood in our model frameworks. We have added a discussion on this topic. Thank you for pointing out a limitation of our simulation. Although we did not clearly delineate the two errors as you have categorized them, we believe that we were able to capture some of that information. In models A and B, there was only a single performance error (difference between performance and desired trajectory). However, in model C, we had two different performance errors that drive the two networks: difference between performance and desired trajectory to drive the trajectory controller, and the difference between performance and the expected output to drive the plant estimator. Using your terminology, we believe that the trajectory error can provide information about self-generated errors, and the plant estimation error can give us information about externally generated performance errors. But since our models were not meant to simulate proprioceptive tasks, we do believe that further adjustments to the models will be needed to properly simulate proprioceptive tasks. A discussion has been added to the limitations. Instead of adding the link between model predictions on page 4 to Figure 4, we have added a description in the results section. Since the experiments were not yet described in page 4, it may be premature to link to Figure 4 at that part of the manuscript. In regards to Figure 2, whether the desired trajectory generator was included in the controller block was only a matter of notation. We chose not to include that in the controller block because no learning occurred in the desired trajectory generation as the desired trajectory was programmed to be a straight line with bell shape velocity from start to end point. The learning network blocks performed the computation for the neural networks described in paragraph 2 and 3 of “Trajectory generator” section of the Simulations. We have updated Figure 2 to better indicate the environment adaptation module. In the text, we have combined the equations for online correction into one equation causing the confusion of the equation mismatch. We have changed the text to reflect the same notations used in the figure. Page 6: Thank you for pointing out the missing c, which has been corrected. The i and j are dummy variables for the summations, we have added clarification for this in the text. The learning rule for P was shown as the equation with delta P. The equation indicated the output has been added for clarity. Regarding the random order of experiments, we did not believe that the repeated exposure of the adaptation task changed subject behavior as the experiment was relatively short compared to most adaptation studies (20 trials per block). The randomization was used to safe guard against any potential adaptation towards the end of the experiment. After the experiment, we did not see any overall patterns of behavior change according to experimental order. The cursor was visible to participants under all experimental conditions during the reach. For the purpose of our study, the intended movement and the target position is essentially the same as long as the subjects were following our instructions. As you pointed out, there are circumstances where there may be disagreements between the two terms such as in Mazzoni and Krakauer paper where they examined the use of explicit strategies. I would argue that Mazzoni and Krakauer were essentially giving their participants another “invisible” target to aim for as we did for our experiment 4, and in that case, the new invisible target becomes the intended movement. But we agree that the particular sentence in question was poorly structured and had been rewritten for clarity. As requested by both reviewers, we performed additional statistical analysis and they are reported in the text and in the new figures 5 and 7." } ] }, { "id": "5806", "date": "09 Sep 2014", "name": "Rajiv Ranganathan", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary:The goal of this paper by Chu and Sanger is to examine the hypothesis that learning mechanisms that adapt to altered environments (like visuomotor rotation) is different from learning mechanisms that are required for improvement of motor skills. Using both experiments and model simulations, the authors make the argument that there are indeed two learning mechanisms -- a learning mechanism for each task (which accounts for the specificity of learning of different skills) but a single learning mechanism for learning the environment (which accounts for generalization within a single environment).The topic addressed in the paper is of great interest to readers in the motor control community. Overall the paper is well-written and the experiments/modeling are well done.Major concerns:The paper does not seem to make reference to a very similar idea of “model-based” and “model-free” learning (Huang et al., 2011, Haith & Krakauer, 2013). Those papers make almost the exact same distinction where model-based learning accounts for tasks like adaptation, whereas model-free mechanisms account for “skill learning” tasks. It would be useful for the authors to look at this distinction and discuss these ideas in relation to the current paper. Huang et al. (2011) also used a similar paradigm to Experiment 3 to show that practicing in an “opposite” visuomotor rotation could lead to savings (faster re-learning) if the hand-direction was the same (due to model-free learning). Therefore, the authors may want to use caution before making statements such as “adaptation to an altered environment is not useful for training task performance”. While the statistics are used in a “within-subject” kind of way with the z-statistic, I have a couple of concerns:The sample standard deviation is estimated from only 10 trials - therefore the use of a t-distribution may be more appropriate than a z-distribution. The sign of the after-effect is masked by this analysis – i.e. both undershoots and overshoots would count as being significant since only the absolute value is being taken into account. It would therefore be useful to do a typical \"between-subjects\" analysis (like a paired t-test) which would also help the reader know the actual magnitudes of the after-effects across the 4 experiments (and not merely whether they were significant or not). Minor:I do not understand how the two stripes are representing “West”. Does one stripe represent West in visual space, and the other in hand space? If so, how did the participants distinguish the two stripes? Also it seems that in Figure 3B, the middle panel “Interference:B1” has the directions marked incorrectly. Figure 2 is a little hard to digest and has repetition of the same blocks across multiple panels – it would help the readers if the authors could omit the equations (or use them just once if they are repeated) so that readers could clearly grasp the critical differences between the 3 models. The rationale behind Experiment 4 is a little unclear to me. What do the authors mean when they say “we had to change the visual display”(do they mean target position?). There also seems to some discrepancy between Figure 3D and the table in Figure 3 (e.g. figure 3D should show Baseline \"A2\" and Readaptation A2, not A1). Also the titles for each experiment condition in Figures 3A-D are all labeled the same \"Baselne A1 Interference B1 and Re-Adaptation A1\". The lesser strength of effects in the 90° experiments could be explained by different strategies used in small versus large visuomotor rotations (Abeele & Bock, 2001). The ideas in the paper would also be helped by reference to older ideas of “task and practice specificity” of motor skills (Henry, 1959) – which suggest poor generalization of motor skills. It would help to line up the experimental data in Figure 5 alongside the model predictions in Figure 4 so that it is easier to identify the best predicting model. Why is there a difference in the learning rate between the 3 models during adaptation to the first task (Figure 4, Experiment 1 trials 1-20)? Is it a fair comparison among the 3 models if the final performance of all models is not equivalent after this first block? Since Experiment 3 is the real important finding that confirms the author's model of motor learning, it would help to show in a Figure the fact that participants actually increase the error in the first few trials (currently it is not possible to see this clearly because all 60 trials are all compressed into the figure).", "responses": [ { "c_id": "1104", "date": "10 Dec 2014", "name": "Virginia Chu", "role": "Author Response", "response": "Thank you for your suggestion on additional discussion areas and statistical analysis. We have addressed your comments in our second version with the details highlighted below.Thank you for your suggestion with the discussion on model-based and model free learning models, we have added a discussion on this topic in the manuscript. Regarding the use of z-statistics as a measure in our study, we agree that this is not a conventional way of using this measure. What we call “z” is using a measure similar to the z-statistics, since our sample size was 1 (aftereffect trial) to be compared with a population size of 10 (baseline). In using a z-statistics-like measure, this allows us to use the baseline mean and standard deviation of the baseline trials as the population mean and standard deviation. However, if we use the t-statistics, it will require that we have the sample’s standard deviation (which is 0 for a sample size of 1). Therefore, we chose to use the z-statistics as the basis of for our statistical measure. In order to show the sign of the after effect for readers to examine, we have changed the reported z value to be without the absolute value, and use \|z\| for statistical comparison. As shown in the new Table 1 and 2, only 3 values were affected, all insignificant z values for Experiment 2. As requested by both reviewers, regarding information for typical “between-subject” analysis, we performed additional statistical analysis and they are reported in the text and in the new figures 5 and 7. Thank you for pointing out the error in the notation in figure 3B. We have corrected that. The 2 stripes were used as 1 symbol. They are together used as an indicator for changes in the rotation environment. They were deliberately vague so that the participants would notice a change, but cannot clearly determine the amount of visual rotation. We made Figure 2 simpler and changed the shading of the border markings to make the models easier to read. Figure 3 has been corrected. We apologize for the mislabeling. We have also clarified the rationale for experiment 4 to make things clearer. Thank you for pointing out two relevant references (Abeele & Bock, 2001) and (Henry, 1959). Unfortunately, we were not able to locate (Henry, 1959). Were you by chance referring to (Henry, 1958/1968)? We have added a discussion on the topics in the introduction and discussion. We agree that comparison between Figure 4 and 5 is important in the identification of the best predicting model. Unfortunately, we feel that putting the subject data from Figure 5 within Figure 4 would make the figures too small to give. Instead, we re-arranged the order of the graphs in Figure 5, to facilitate comparison between the two figures. The model simulations are meant for qualitative comparisons. Thank you for pointing out that the learning rate and final performance in the end of the first block was not the same. Our primary focus was to keep the model components to be as consistent as possible across the 3 models, so that the only difference between the models is the model structure. As an example, each learning network in the controller and the time update in the online correction (Figure 2) for all 3 models used the same model parameters. The differences observed in the simulation results, simply result from the difference in model structure. We felt that this was a more important comparison to examine from the simulation results, rather than tuning the model parameters so that the learning rates and final performance to be equal. It is important to note that the difference in simulation results stemmed simply from a difference in model structure using the exact same model components and parameters. Model C appear to have higher learning rate because it has an additional learning component (environment adaptation controller) compared to model A and B. Model A and B could be made to have the same learning rate and final performance in the baseline block by tuning λ, the learning rate of the system (using a faster rate). But this would not change the pattern of results we see in the simulation, and it is more important to us that the model parameters were kept the same.In order to demonstrate the errors in Experiment 3 more clearly, the trajectory errors were shown in Figure 6, where the last trial of baseline and the first trial of the remaining 2 blocks were shown. Further, the errors were also show numerically in Table 1. With the new Figure 5E and Figure 7E, the group means of the errors in the first trial in state C are now shown clearly in a bar chart form." } ] } ]	1	https://f1000research.com/articles/3-72
https://f1000research.com/articles/3-255/v1	28 Oct 14	{ "type": "Case Report", "title": "Case Report: Mammary and rectal metastases from an ovarian cancer: report of two cases and review of literature", "authors": [ "Mounia Amzerin", "Camilo Garcia", "Claudia Stanciu", "Isabelle Veys", "Ahmad Awada", "Hassan Errihani", "Andrea Gombos", "Camilo Garcia", "Claudia Stanciu", "Isabelle Veys", "Ahmad Awada", "Hassan Errihani", "Andrea Gombos" ], "abstract": "In this paper we report two interesting cases of metastatic ovarian cancer. The first case is a patient who developed rectal and breast metastases mimicking an inflammatory breast cancer. In the second case, subclinical breast and axillary lymph node metastases were revealed by PET/CT. Metastases in the breast originating from solid tumors are extremely rare. The ovarian primitive is the fourth most common origin. The occurrence of breast metastasis is associated with an advanced disease and a poor prognosis. Their incidence is increasing since they are found more often due to better imaging techniques and to better treatment that, accordingly, improve patients’ survival. Thus, unusual sites of metastases are more and more reported. Indeed, some authors reported the occurrence of colorectal metastases from ovarian cancer. However, they remain much less frequent.", "keywords": [ "In November 2010", "a 61 year old Moroccan housewife was diagnosed with a stage IV poorly differentiated serous ovarian adenocarcinoma (peritoneal", "mediastinal and retroperitoneal lymph node metastasis). The patient had a medical history of diabetic neuropathy and hypertension. Her family history noted a sister and a niece who were respectively diagnosed with pancreatic cancer and breast cancer. A retroperitoneal lymph node biopsy was performed to obtain tumor tissue for histological diagnosis. Given the spread of the disease it was decided to administer chemotherapy first. Since she had a contraindication to taxane-based regimens because of her peripheral neuropathy", "the patient received 3 cycles of Cyclophosphamide 600 mg/m2 and Carboplatin AUC 5 in the first cycle and AUC6 in the subsequent cycles", "every three weeks. The response assessment with PET-FDG after the third cycle showed partial response. One and a half months after the third cycle of chemotherapy", "she underwent debulking surgery. The treatment was completed by two additional cycles of chemotherapy of the same combination." ], "content": "Case 1\n\nIn November 2010, a 61 year old Moroccan housewife was diagnosed with a stage IV poorly differentiated serous ovarian adenocarcinoma (peritoneal, mediastinal and retroperitoneal lymph node metastasis). The patient had a medical history of diabetic neuropathy and hypertension. Her family history noted a sister and a niece who were respectively diagnosed with pancreatic cancer and breast cancer. A retroperitoneal lymph node biopsy was performed to obtain tumor tissue for histological diagnosis. Given the spread of the disease it was decided to administer chemotherapy first. Since she had a contraindication to taxane-based regimens because of her peripheral neuropathy, the patient received 3 cycles of Cyclophosphamide 600 mg/m2 and Carboplatin AUC 5 in the first cycle and AUC6 in the subsequent cycles, every three weeks. The response assessment with PET-FDG after the third cycle showed partial response. One and a half months after the third cycle of chemotherapy, she underwent debulking surgery. The treatment was completed by two additional cycles of chemotherapy of the same combination.\n\nThree months after the end of treatment, a CT scan showed progressive disease in the mediastinal and abdominal lymph nodes. The patient received Liposomal Doxorubicin 40 mg/m2 q4w, as second line chemotherapy. The response assessment after three cycles showed disease progression. Since the patient had been asymptomatic, it was decided to wait and see.\n\nFour months later, in January 2012, the patient presented with skin erythema and edema of the right breast. The clinical examination found ipsilateral supraclavicular lymph node swelling. The patient also complained of a diarrhea, which was resistant to standard treatments. Breast MRI showed breast and chest edema with multiple non-specific contrast uptakes giving the aspect of a homogeneous enhancement. The patient underwent sigmoidoscopy that revealed extended ulcerations of the lining of the rectum (Figure 1A). The breast and rectal biopsies showed a positive staining of PAX8 by immunohistochemistry (Figure 1B). Both lead to a diagnosis of metastasis from the known serous ovarian neoplasia. The patient was treated by topotecan as third line chemotherapy Topotecan 4 mg/m2 days 1, 8, 15; every 28 days. Unfortunately the disease progressed dramatically after two cycles. The patient died in March 2012, 18 months after the initial diagnosis, 3 months after the diagnosis of the breast metastasis.\n\nUlceration in the lining of the rectum corresponding to metastases from the ovarian primary.\n\n20× magnification. Positive staining PAX8 on immunohistochemistry: Nuclear staining dark brown in tumor cells of ovarian origin within the mammary stroma stained blue.\n\n\nCase 2\n\nA 52 year old Caucasian patient was diagnosed with a cyst of the right ovary. She had a medical history of a herniated disc and cholecystectomy. She had no family history of cancer. During an exploratory laparoscopy, a peritoneal carcinomatosis was found. The patient underwent a suboptimal debulking surgery consisting of hysterectomy and bilateral salpingo-oophorectomy. The histopathological examination showed a serous papillary adenocarcinoma of the ovary, classified as at least FIGO stage IIIc. She was then treated with 3 cycles of combination chemotherapy consisting of Paclitaxel 175 mg/m2 and Carboplatin AUC 6 q3w. The patient underwent a second surgery one month after the third cycle of chemotherapy, to perform omentectomy and lumboaortic node dissection. From 25 resected lymph nodes, 14 were positive for tumor cells. The patient received three additional cycles of the same combination of chemotherapy.\n\nSeventeen months later, the patient consulted her doctor for nausea and abdominal pain. The physical examination was strictly normal. The blood test showed an increased Ca125 tumor marker. The PET/FDG revealed recurrence of the cancer in the peritoneum. Moreover, it showed a Fludeoxyglucose (FDG) uptake at PET-CT in the right breast and the ipsilateral axillary lymph node suggesting a primary breast cancer (Figure 2A). Both ultrasound and MRI confirmed multiple lesions in the right breast. An ultrasound guided biopsy showed metastasis from ovarian cancer corresponding to a PAX8 overexpression on the histological examination (Figure 2B). The patient received a second line chemotherapy consisting of Gemcitabine 1000 mg/m2 d1, d8 and Carboplatin AUC 4 d1 q3w. After six cycles, a CT scan showed partial response according to RECIST criteria1 whereas metabolic assessment by PET/FDG-CT revealed a stable disease. The reassessment three months after the end of chemotherapy showed progressive disease. The patient received Liposomal Doxorubicin 40 mg/m2 q4w. After six cycles, the disease progressed. The patient received third line chemotherapy consisting of weekly Paclitaxel 80 mg/m2 d1, d8, d15 q4w. The last assessment of tumor response to Paclitaxel in May 2014 showed stable disease.\n\nFused FDG PET/CT images showing an intense focal FDG uptake in the right breast coinciding with a dense breast nodule on CT images. The hot iron FDG PET scale represents intensity of FDG uptake, varying from black at the weakest intensity to white at the strongest.\n\n4× magnification. Positive staining PAX8 on immunohistochemistry: Positive staining PAX8 on immunohistochemistry: Nuclear staining dark brown in tumor cells of ovarian origin within the mammary stroma stained blue.\n\n\nDiscussion\n\nBreast metastases from a non-mammary origin remain anecdotal. Some retrospective studies of patients with metastatic cancers from different primaries, report an incidence ranging from 0.1 to 1.3%2–4. Over a period of 20 years, Delair et al. recorded only 85 cases5.\n\nMost often, metastases in the breast originate from lymphomas and melanomas. Among the gynecological primitives, ovarian serous carcinoma is the most frequent5,8, which is the case in our two patients. Samples from 825 breast cancers were studied by high throughput sequencing techniques and compared with basal like breast cancer, using data originating from the Cancer genome Atlas9. Interestingly, it was discovered that basal-like tumors share the same driving mutations, i.e. they are genetically similar to high grade serous ovarian carcinomas9. We are not sure if this could explain the fact that serous ovarian carcinoma is the one that, in comparison with other gynecological primaries, is the most frequently responsible for metastasis in the breast. Unfortunately, in the case of our patients, we were not able to perform deep gene sequencing analysis since we didn’t have archived frozen tissue.\n\nFewer than 50 similar cases have been reported since 1907, when A. Sitzenfrey first described a patient diagnosed with breast metastases of ovarian origin10,11. The incidence of extra mammary metastases is increasing due to the improvements in treatment leading to longer survival. Thus, unusual sites of metastases are more and more reported. In addition, the frequent use of modern images techniques like PET-CT leads to the detection of subclinical lesions. When the PET-CT was used for the work-up among patients with FIGO stage IIC-IV ovarian cancer, a supradiaphragmatic lymph node disease was detected in 67% of cases even if conventional imaging had showed no metastasis12. This was also the case in both patients described here.\n\nThe most frequent metastatic sites from ovarian cancer are the peritoneum, omentum, colon, lungs and lymph nodes. The breast metastases are very uncommon. They can occur by both hematogenous and lymphatic spread13.\n\nWhen breast metastases from an extra-mammary origin occur, which is exceptional, they are generally diagnosed in patients with a known metastatic disease. Thus, the diagnosis of mammary metastasis from an ovarian origin is often evoked, especially when their occurrence is metachronous. When the diagnosis of a breast tumor and an ovarian primary are made simultaneously, a breast biopsy should be performed in order to exclude a primary breast cancer diagnosis. That is because the association of two distinct primaries is not rare especially for patients carrying BRCA1/2 mutations. However, all clinical presentations are possible. A case of breast metastases announcing an ovarian cancer has also been previously reported14.\n\nUnlike primary breast cancer, extra mammary metastases are usually superficial, mobile, well circumscribed and painless10,15. Although, they can have a clinical presentation mimicking an inflammatory breast cancer, these cases are even rarer. To our knowledge, only seven cases of metastatic ovarian cancer to the breast have been described so far13.\n\nThe mean interval between the diagnosis of ovarian cancer and the breast metastasis (if it occurs) is between 2 and 3 years8,16. Although shorter term has also been reported13. Our two patients developed secondary breast disease during the second year after diagnosis.\n\nImaging techniques could help to diagnose breast metastases. On a mammography, they are usually well circumscribed and dense without spiculations or microcalcifications13. However, serous histology can be associated with microcalcifications so differential diagnosis can sometimes be difficult8,17. However, these imaging techniques are unable to formally distinguish a primary breast cancer from a metastasis.\n\nA biopsy remains the only way to confirm the diagnosis. The classical histopathological examination with Hematoxylin and eosin stain could be inconclusive, since both ovarian and breast cancers have papillary, glandular or solid architecture. Standard immunohistochemical analysis is often unhelpful, because both ovarian and breast primary tumors are usually CK7 positive and CK20 negative. In addition, both cancers could express or not, estrogen and/or progesterone receptors. So far, PAX8 is the only known marker that can reliably make the differential diagnosis between breast cancer and ovarian cancer, since gynecological cancers are PAX8-positive whereas breast cancers are PAX8-negative18. Wilm’s Tumor 1 Receptor WT1 has also been described as an interesting marker to make the differential diagnosis between a metastasis from ovarian origin and a breast primary, although some breast cancers have been found positive for WT118,19. For our patients, the pathologist used PAX8-staining to confirm the diagnosis of metastasis from ovarian origin.\n\nDifferential diagnosis is crucial in order to define the appropriate treatment for the patient. If the diagnosis is primary breast cancer, then the treatment would be a combination of surgery, chemotherapy, biological agents (such as anti-Her2 therapy her2 positive tumors, and antiangiogenic therapy), hormonal therapy if the cancer expresses hormonal (estrogen, progesterone) receptors and/or radiotherapy if indicated. In the case of breast metastases, surgery should not to be the first option, since in metastatic ovarian cancer the chemotherapy is the main course of treatment.\n\nUnfortunately, breast metastasis of an ovarian cancer indicates an extensive disease. The prognosis is poor. Previous studies report survivals ranging between 13 days and 3.5 years10. However, rare cases of longer overall survival have also been observed10,14. Our first patient died only 2 months after the diagnosis of the breast metastasis.\n\nFinally, the case of our first patient is also interesting regarding the presence of histologically confirmed rectal metastases. Only 5 cases of colorectal metastases from an ovarian cancer have been reported, mainly in the clear cell carcinoma subtype20–24. Thus, in this paper, we have described the sixth case and the first case with both breast-and rectal metastases from a serous papillary ovarian carcinoma.\n\n\nConclusion\n\nTo our knowledge, this is the first case reported in literature that describes both rectal- and breast metastases mimicking an inflammatory breast cancer, that derived from a serous papillary ovarian cancer. Our second case illustrates the role of PET-CT in detecting subclinical metastases, which leads to an increase in the diagnosis of uncommon sites of secondary dissemination of ovarian cancer. The differential diagnosis between a primary and a secondary breast cancer is crucial to provide the appropriate treatment. Unfortunately, the occurrence of breast metastases in an ovarian carcinoma is linked to an extensive disease and a poor prognosis.\n\n\nPatient consent\n\nThe consents of the first patient’s daughter and of the second patient were obtained before submitting to publication.", "appendix": "Author contributions\n\n\n\nAmzerin, M. and Gombos, A. performed a literature review, the composition of this case report and manuscript writing.\n\nStanciu, C. and Garcia, C. were charged with the pathological and metabolic imaging studies respectively.\n\nVeys, I., Gombos, A., Errihani, H., and Awada, A. corrected and approved the final version of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in the funding of this work.\n\n\nAcknowledgments\n\nThe authors would like to thank Pr Chaker El Amrani for editorial support.\n\n\nReferences\n\nEisenhauera EA, Therasseb P, Bogaertsc J, et al.: New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1). Eur J Cancer. 2009; 45(2): 228–247. PubMed Abstract \| Publisher Full Text\n\nSurov A, Fiedler E, Holzhausen HJ, et al.: Metastases to the breast from non-mammary malignancies: primary tumors, prevalence, clinical signs, and radiological features. Acad Radiol. 2011; 18(5): 565–574. PubMed Abstract \| Publisher Full Text\n\nWHO Classification of tumours of the Breast -international Agency for Research on Cancer -4th edition, Lyon, 2012.\n\nAFIP Atlas of Tumor Pathology series 4 - tumors of the Mammary Gland Washington, DC. 2009.\n\nBertella L, Kaye J, Perry NM, et al.: Metastases to the breast revisited: radiological-histopathological correlation. Clin Radiol. 2003; 58(7): 524–531. PubMed Abstract \| Publisher Full Text\n\nDelair DF, Corben AD, Catalano JP, et al.: Non-Mammary metastases to the breast and axilla: a study of 85 cases. Mod Pathol. 2013; 26(3): 343–349. PubMed Abstract \| Publisher Full Text\n\nVizcaino I, Torregrosa A, Higueras V, et al.: Metastasis to the breast from extramammary malignancies: a report of four cases and a review of literature. Eur Radiol. 2001; 11(9): 1659–1665. PubMed Abstract \| Publisher Full Text\n\nMoore DH, Wilson DK, Hurteau JA, et al.: Gynecologic cancers metastatic to the breast. J Am Coll Surg. 1998; 187(2): 178–81. PubMed Abstract \| Publisher Full Text\n\nCancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours. Nature. 2012; 490(7418): 61–70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaykal C: Breast and ovarian carcinoma in the same patient, metastasis or dual primaries. Turk J Cancer. 2007; 37(1): 27–30. Reference Source\n\nSitzenfrey A: Mammakarzinom zwei jahre nach abdominal radikaloperation wegen doppelseitigen carcinoma ovarii. Prag Med Wochenschr. 1907; 32: 221–35.\n\nHynninen J, Auranen A, Carpén O, et al.: FDG PET/CT in staging of advanced epithelial ovarian cancer: Frequency of supradiaphragmatic lymph node metastasis challenges the traditional pattern of disease spread. Gynecol Oncol. 2012; 126(1): 64–68. PubMed Abstract \| Publisher Full Text\n\nKlein RL, Brown AR, Gomez-Castro CM, et al.: Ovarian Cancer Metastatic to the Breast Presenting as Inflammatory Breast Cancer: A Case Report and Literature Review. J Cancer. 2010; 1: 27–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchneuber SE, Scholz HS, Regitnig P, et al.: Breast metastasis 56 months before the diagnosis of primary ovarian cancer: a case study. Anticancer Res. 2008; 28(5B): 3047–3050. PubMed Abstract\n\nPaulus DD, Libshitz HI: Metastasis to the breast. Radiol Clin North Am. 1982; 20(3): 561–8. PubMed Abstract\n\nLaifer S, Buscma J, Parmley TH, et al.: Ovarian Cancer metastatic to the breast. Gynecol Oncol. 1986; 24(1): 97–102. PubMed Abstract \| Publisher Full Text\n\nChaignaud B, Hall TJ, Powers C, et al.: Diagnosis and natural history of extramammary tumors metastatic to the breast. J Am Coll Surg. 1994; 179(1): 49–53. PubMed Abstract\n\nNonaka D, Chiriboga L, Soslow RA: Expression of pax8 as a useful marker in distinguishing ovarian carcinomas from mammary carcinomas. Am J Surg Pathol. 2008; 32(10): 1566–71. PubMed Abstract \| Publisher Full Text\n\nZhao L, Guo M, Sneige N, et al.: Value of PAX8 and WT1 immunostaining in confirming the ovarian origin of metastatic carcinoma in serous effusion specimens. Am J Clin Pathol. 2012; 137(2): 304–309. PubMed Abstract \| Publisher Full Text\n\nTrastour C, Rahili A, Schumacker A, et al.: Haematogenous rectal metastasis 20 years after removal of epithelial ovarian cancer. Gynecol Oncol. 2004; 94(2): 584–8. PubMed Abstract \| Publisher Full Text\n\nZighelboim I, Broaddus R, Ramirez PT: Atypical sigmoid metastasis from a high-grade mixed adenocarcinoma of the ovary. Gynecol Oncol. 2004; 94(3): 850–3. PubMed Abstract \| Publisher Full Text\n\nDowdy SC, Pfeifer EA, Longcope DC, et al.: Unusual recurrence of ovarian carcinoma 9 years after initial diagnosis. Gynecol Oncol. 1999; 74(3): 495–8. PubMed Abstract \| Publisher Full Text\n\nLee EJ, Deavers MT, Hughes JI, et al.: Metastasis to sigmoid colon mucosa and submucosa from serous borderline ovarian tumor: response to hormone therapy. Int J Gynecol Cancer. 2006; 16(Suppl 1): 295–9. PubMed Abstract \| Publisher Full Text\n\nBijek JH, Ehnart N, Mathevet P: Hematogenous dissemination in epithelial ovarian cancer: Case report. J Gynecol Obstet Biol Reprod (Paris). 2011; 40(5): 465–8. PubMed Abstract \| Publisher Full Text" }	[ { "id": "6546", "date": "31 Oct 2014", "name": "Daniel Vorobiof", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nInteresting case reports. I agree with the authors that as imaging is becoming more accurate and available, unusual sites of metastatic disease are diagnosed, contributing to our understanding of the evolution of different malignant diseases.", "responses": [] }, { "id": "6791", "date": "01 Dec 2014", "name": "Véronique D'Hondt", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTwo interesting cases of unusual evolution of ovarian carcinoma.Well written paper with a good discussion and a large review of literature.Congratulations to the authors!Minor comments:page 2; case 2; 12th line: lomboaortic instead of lumboaorticpage 3; discussion; end of 2nd paragraph: we haven't archivedpage 3; discussion; 3rd paragraph: modern imaging techniquespage 4; 4th paragraph: help diagnosing", "responses": [] } ]	1	https://f1000research.com/articles/3-255
https://f1000research.com/articles/3-297/v1	08 Dec 14	{ "type": "Case Report", "title": "Case Report: A case report of Moyamoya disease in a 36 year old African American woman", "authors": [ "Rohit Kumar Gudepu", "Mohtashim A. Qureshi", "Ihtesham A. Qureshi", "Lakshman Rao", "Mohtashim A. Qureshi", "Ihtesham A. Qureshi", "Lakshman Rao" ], "abstract": "Moyamoya is a rare idiopathic progressive vaso-occlusive disease characterized by irreversible condition of main blood vessels to the brain as they enter into the skull. We present a case of 36 year old African American female presenting to the Out Patient Clinic with headache which were on and off for 4-6 months and did not relieve on routine medical therapy. It was associated with weakness on right side for last few days. The patient was investigated with CT Angiogram, diagnosed as Moyamoya disease and operated. She has been followed up for the last 5 years and the patient has not complained of any headaches or focal neurological symptoms.", "keywords": [ "Moyamoya disease is a progressive occlusive condition involving cerebral vessels which includes mainly", "stenosis of distal internal carotid arteries on both sides and anterior and middle cerebral arteries thereby leading to development of collateral vessels to compensate for the occlusion. Moyamoya is a rare disease with reported incidence of 0.086 per 100", "000 population1. Though", "originally considered to affect predominantly persons of Asian heritage it is now seen throughout the world in people of many ethnic backgrounds2", "3. There are nearly twice as many female patients as male patients4", "9", "10. Moyamoya disease usually is associated with condition like Sickle cell disease", "Down’s Syndrome and Neurofibromatosis-14–7." ], "content": "Introduction\n\nMoyamoya disease is a progressive occlusive condition involving cerebral vessels which includes mainly, stenosis of distal internal carotid arteries on both sides and anterior and middle cerebral arteries thereby leading to development of collateral vessels to compensate for the occlusion. Moyamoya is a rare disease with reported incidence of 0.086 per 100,000 population1. Though, originally considered to affect predominantly persons of Asian heritage it is now seen throughout the world in people of many ethnic backgrounds2,3. There are nearly twice as many female patients as male patients4,9,10. Moyamoya disease usually is associated with condition like Sickle cell disease, Down’s Syndrome and Neurofibromatosis-14–7.\n\n\nCase description\n\nA 36 year old African American female presented to the Out Patient Office with headache which was on and off for last 4–6 months. The headache presented was of piercing type with moderate intensity not relieved by butalbital, acetaminophen. It was associated with weakness on right hand side for last few days, which evolved into dizziness and speech difficulty. There was no associated fever, night sweats, loss of consciousness, vomiting, ataxia, photophobia, tingling, numbness, difficulty swallowing, difficulty hearing, neck stiffness, vision problems. Her family history was significant for hypertension, diabetes and coronary artery disease. General physical examination showed no abnormalities. She is obese with BMI of 36.4; Vitals include [Heart Rate = 93/min, Blood Pressure = 165/75 mm Hg, Temp. = 36.67°C, Resp. Rate = 18/min]. Neurological examination showed intact 2–12 cranial nerve function, motor strength grossly intact with strength 3/5 on the right side of hand. No pronator drift, normal muscular tone, and gait was normal. Laboratory investigations, complete blood count, electroencephalogram and echocardiogram were normal.\n\nA CT angiogram head with contrast shows one sagittal [Figure 1] and two axial views [Figure 2, Figure 3] at the level of the lateral ventricle and Circle of Willis, demonstrate mild proliferation of collateral vessels emanating from the distal internal carotid artery particularly on the left side. In addition, the visualized portion of the distal internal carotid artery and M1 segment of the left middle cerebral arteries appeared somewhat diminutive in caliber. Findings were generally compatible with progressive vascular occlusive process which can be seen in Moyamoya disease.\n\nIn addition, the visualized portion of the distal internal carotid artery and M1 segment of the left middle cerebral artery appear somewhat diminutive in caliber.\n\nIn addition, the visualized portion of the distal internal carotid artery and M1 segment of the left middle cerebral artery appear somewhat diminutive in caliber.\n\nSince the patient was refractory to the pain medications, surgical revascularization was done and the procedure involved left superficial temporal artery-left middle cerebral artery bypass without any complications. Following surgery, she was prescribed 81mg aspirin and discharged home and was followed up initially after surgery once every three months for one year followed by every 6 months for next four years. She did not complain of any headaches or focal neurological symptoms.\n\n\nDiscussion\n\nMoyamoya disease is a chronic, progressive occlusion of the Circle of Willis arteries that leads to the development of characteristic collateral vessels seen on imaging, particularly cerebral angiography8. Moyamoya was originally considered to affect predominantly persons of Asian heritage but has now been observed throughout the world. The incidence peaks lie within two age groups: children who are 5 years old and adults in their mid 40’s9–12. Moyamoya disease is rarely seen in the African American population. Uchino et al.1 found only 27 African Americans with Moyamoya disease in the states of California and Washington. According to the year 2000 US Census population, only 44 African Americans were diagnosed with Moyamoya every year8.\n\nMoyamoya usually presents with recurrent headaches and is migraine like in quality and refractory to medical therapies. Being a chronic progressive occlusive condition it causes stenosis of intracranial internal carotid arteries and their proximal branches causing reduced blood supply to the anterior surface of brain, thereby leading to the formation of collaterals near the apex of carotids which look like “Puffs of Smoke” known as Moyamoya in Japanese13. The process of blockage, once it begins, tends to continue despite any known medical management unless treated with surgery14.\n\nThough MRI angiography is used to confirm the diagnosis and to see the anatomy of the vessels involved, CT angiography can also be used to see intracranial stenoses suggesting Moyamoya. Thus, CT angiography can be considered when MRI is not readily available and a diagnosis of cerebral occlusive vasculopathy is being considered15. Since surgery is the only viable option, revasularization procedures are gaining importance as a primary treatment for Moyamoya, given the poor response to medical therapy and documented success of surgery17.\n\nA good option for adult symptomatic patients is superficial temporal artery-middle cerebral artery bypass or middle meningeal artery to middle cerebral artery bypass18–21. Patients have reported to have 96% probability of remaining stroke free over the subsequent five years4,16. The present patient had a successful surgical treatment without any recurrence of symptoms. Nevertheless, the outcome can be predicted based on the neurological status at the time of treatment, more than the patient’s age4. Hence, it is necessary for early diagnosis of the condition followed by surgical intervention therapy. The estimated rate of symptomatic progression is only 2.6% after surgery according to a meta-analysis involving 1156 patients17.\n\nThis case highlights the importance of considering Moyamoya disease as one of the differentials while dealing with patients with recurrent headaches who are in their third/fourth decade of life and children around 5 years old in whom headache is not relieved on routine medical treatment. It also emphasizes on the rare presentation among African American population and use of CT angiography as an alternative diagnostic imaging tool for diagnosing Moyamoya in cases of MRI non-availability.\n\n\nPatient consent\n\nInformed written consent for publication of clinical details was obtained from the patient.", "appendix": "Author contributions\n\n\n\nRG, MQ, IQ have performed literature review and manuscript writing. LR helped to make the diagnosis. All the authors approved the final version of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nUchino K, Johnston SC, Becker KJ, et al.: Moyamoya disease in Washington State and California. Neurology. 2005; 65(6): 956–8. PubMed Abstract \| Publisher Full Text\n\nCaldarelli M, Di Rocco C, Gaglini P: Surgical treatment of moyamoya disease in pediatric age. J Neurosurg Sci. 2001; 45(2): 83–91. PubMed Abstract\n\nSuzuki J, Kodama N: Moyamoya disease--a review. Stroke. 1983; 14(1): 104–9. PubMed Abstract \| Publisher Full Text\n\nScott RM, Smith JL, Robertson RL, et al.: Long-term outcome in children with moyamoya syndrome after cranial revascularization by pial synangiosis. J Neurosurg. 2004; 100(2 Suppl Pediatrics): 142–9. PubMed Abstract \| Publisher Full Text\n\nJea A, Smith ER, Robertson R, et al.: Moyamoya syndrome associated with Down syndrom: outcome after surgical revascularization. Pediatrics. 2005; 116(5): e694–e701. PubMed Abstract \| Publisher Full Text\n\nHankinson TC, Bohman LE, Heyer G, et al.: Surgical treatment of moyamoya syndrome in patients with sickle cell anemia: outcome following encephaloduroarteriosynangiosis. J Neurosurg Pediatr. 2008; 1(3): 211–6. PubMed Abstract \| Publisher Full Text\n\nUllrich NJ, Robertson R, Kinnamon DD, et al.: Moyamoya following cranial irradiation for primary brain tumors in children. Neurology. 2007; 68(12): 932–8. PubMed Abstract \| Publisher Full Text\n\nJanda PH, Bellew JG, Veerappan V: Moyamoya disease: case report and literature review. J Am Osteopath Assoc. 2009; 109(10): 547–53. PubMed Abstract\n\nBaba T, Houkin K, Kuroda S: Novel epidemiological features of moyamoya disease. J Neurol Neurosurg Psychiatry. 2008; 79(8): 900–4. PubMed Abstract \| Publisher Full Text\n\nWakai K, Tamakoshi A, Ikezaki K, et al.: Epidemiological features of moyamoya disease in Japan: findings from a nationwide survey. Clin Neurol Neurosurg. 1997; 9(Suppl 2): S1–S5. PubMed Abstract \| Publisher Full Text\n\nHan DH, Nam DH, Oh CW: Moyamoya disease in adults: characteristics of clinical presentation and outcome after encephalo-duro-arterio-synangiosis. Clin Neurol Neurosurg. 1997; 99(Suppl 2): S151–S155. PubMed Abstract \| Publisher Full Text\n\nHan DH, Kwon OK, Byun BJ, et al.: A co-operative study: clinical characteristics of 334 Korean patients with moyamoya disease treated at neurosurgical institutes (1976–1994). The Korean Society for Cerebrovascular Disease. Acta Neurochir (Wien). 2000; 142(11): 1263–73. PubMed Abstract \| Publisher Full Text\n\nFukui M: Guidelines for the diagnosis and treatment of spontaneous occlusion of the circle of Willis (‘moyamoya’ disease). Research Committee on Spontaneous Occlusion of the Circle of Willis (Moyamoya Disease) of the Ministry of Health and Welfare, Japan. Clin Neurol Neurosurg. 1997: 99(Suppl 2): S238–S240. PubMed Abstract \| Publisher Full Text\n\nSmith ER, Smith RM: “Moyamoya syndrome associated with Congenital heart diseases”. Skull Base. 2005; 15(1): 15–26.\n\nScott RM, Smith ER: “Moyamoya disease and moyamoya syndrome”. N Eng J Med. 2009; 360(12): 1226–37. PubMed Abstract \| Publisher Full Text\n\nChoi JU, Kim DS, Kim EY, et al.: Natural history of moyamoya disease: comparison of activity of daily living in surgery and non surgery groups. Clin Neurol Neurosurg. 1997; 99(Suppl 2): S11–S18. PubMed Abstract \| Publisher Full Text\n\nFung LW, Thompson D, Ganesan V: Revascularization surgery for pediatric moyamoya: a review of the literature. Childs Nerv Syst. 2005; 21(5): 358–64. PubMed Abstract \| Publisher Full Text\n\nKuroda S, Ishikawa T, Houkin K, et al.: Incidence and clinical features of disease progression in adult moyamoya disease. Stroke. 2005; 36(10): 2148–2153. PubMed Abstract \| Publisher Full Text\n\nOya S, Tsutsumi K, Ueki K: Adult-onset moyamoya disease with repetitive ischemic attacks successfully treated by superficial temporal-middle cerebral artery bypass--case report. Neurol Med Chir (Tokyo). 2003; 43(3): 138–141. PubMed Abstract \| Publisher Full Text\n\nGolby AJ, Marks MP, Thompson RC, et al.: Direct and combined revascularization in pediatric moyamoya disease. Neurosurgery. 1999; 45(1): 50–58. PubMed Abstract\n\nIwama T, Hashimoto N, Miyaka H, et al.: Direct revascualrization to the anterior cerebral artery territory in patients with moyamoya disease: report of five cases. Neurosurgery. 1998; 42(5): 1157–1161. PubMed Abstract \| Publisher Full Text" }	[ { "id": "6982", "date": "16 Dec 2014", "name": "Bruce Campbell", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAn interesting rare disease case report - NB in Intro states \"Moyamoya disease usually is associated with condition like Sickle cell disease, Down’s Syndrome and Neurofibromatosis\" -whilst all of these associations are correct, the majority of Moyamoya patients do not have one of these conditions.", "responses": [] }, { "id": "7591", "date": "16 Feb 2015", "name": "Johannes Boltze", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript delivers a comprehensive, well-structured and interesting case study on Moya-Moya. The paper is nicely focused and compact, also providing a well-balanced discussion.Apart from agreeing on Dr. Campell’s comment, the only recommendation I have is to avoid repetitive text elements. Particularly, the first few sentences in the second paragraph of the case description are almost identical to the legend of Figure 2 and may therefore require rephrasing.", "responses": [] } ]	1	https://f1000research.com/articles/3-297
https://f1000research.com/articles/3-259/v1	29 Oct 14	{ "type": "Research Note", "title": "Discovery of functional non-coding conserved regions in the α-synuclein gene locus", "authors": [ "Lori Sterling", "Michael Walter", "Dennis Ting", "Birgitt Schüle", "Lori Sterling", "Michael Walter", "Dennis Ting" ], "abstract": "Several single nucleotide polymorphisms (SNPs) and the Rep-1 microsatellite marker of the α-synuclein (SNCA) gene have consistently been shown to be associated with Parkinson’s disease, but the functional relevance is unclear. Based on these findings we hypothesized that conserved cis-regulatory elements in the SNCA genomic region regulate expression of SNCA, and that SNPs in these regions could be functionally modulating the expression of SNCA, thus contributing to neuronal demise and predisposing to Parkinson’s disease.In a pair-wise comparison of a 206kb genomic region encompassing the SNCA gene, we revealed 32 evolutionary conserved DNA sequences between human and mouse. All elements were cloned into reporter vectors and assessed for expression modulation in dual luciferase reporter assays. We found that 11 out of 32 elements exhibited either an enhancement or reduction of the expression of the reporter gene. Three elements upstream of the SNCA gene displayed an approximately 1.5 fold (p<0.009) increase in expression. Of the intronic regions, three showed a 1.5 fold increase and two others indicated a 2 and 2.5 fold increase in expression (p<0.002). Two elements downstream of the SNCA gene showed 1.5 fold and 2.5 fold increase (p<0.0009). One element downstream of SNCA had a reduced expression of the reporter gene of 0.35 fold (p<0.0009) of normal activity.Our results demonstrate that the SNCA gene contains cis-regulatory regions that might regulate the transcription and expression of SNCA. Further studies in disease-relevant tissue types will be important to understand the functional impact of regulatory regions and specific Parkinson’s disease-associated SNPs and its function in the disease process.", "keywords": [ "α-synuclein", "transcriptional regulation", "evolutionary conserved genomic region", "SNCA", "Parkinson’s disease" ], "content": "Introduction\n\nAn emerging hypothesis is gaining increasing interest and is based on the concept that subtle overexpression of α-synuclein (α-syn) over many decades can either predispose or even cause the neurodegenerative changes that characterize Parkinson’s disease (PD). Neurons subjected to higher, non-physiological levels of α-syn might be more likely to be damaged by oligomerization or aggregation of this protein, eventually leading to the formation of α-synuclein-based neuropathological features of the disease1.\n\nIt is now well established that both point mutations and large genomic multiplications of the α-syn (SNCA) gene can cause an autosomal-dominant form of PD2–10. Furthermore, several association studies investigating genetic variants in the SNCA gene have found an increased risk for PD11–19. The finding that both qualitative and quantitative alterations in the SNCA gene are associated with the development of a parkinsonian phenotype indicates that amino acid substitutions as well as overexpression of wild-type α-syn are capable of triggering a clinicopathological process that is very similar to sporadic PD. Nevertheless, the precise mechanisms leading to α-syn-related pathology in sporadic PD in the absence of any α-syn mutations remain elusive.\n\nThe best characterized polymorphism in the SNCA gene is the Rep-1 mixed dinucleotide repeat which has been shown to act as a modulator of SNCA transcription14–16. The DNA binding protein and transcriptional regulator PARP-1 showed specific binding to SNCA-Rep1. These data were confirmed by a transgenic mouse model and demonstrated regulatory translational activity20.\n\nFunctionally, SNCA expression levels in postmortem brains suggest that the Rep-1 allele and SNPs in the 3′ region of the SNCA gene have a significant effect on SNCA mRNA levels in the substantia nigra and the temporal cortex21.\n\nThe promoter region of the SNCA gene has been recently examined in more detail in cancer cell lines and also in rat cortical neurons. Regulatory regions in intron 1 and the 5′ region of exon 1 have been shown to exhibit transcriptional activation22–24 as well as the NACP-Rep-1 region upstream of the SNCA gene14–16,20,25. Several transcription factors have been identified such as PARP-116, GATA26, ZIPRO1, and ZNF21922 to have an effect on regulating the SNCA promoter region.\n\nThere is mounting evidence that SNCA expression levels could be crucial for maintenance and survival of neurons and its misregulation could play a key role in the development of PD. Thus, the importance of thoroughly investigating the SNCA gene to fully understand its cis- and trans-acting elements and factors and for the functional interpretation of the PD-disease associated risk alleles is becoming increasingly clear.\n\nThe goal of this study was to investigate transcriptional regulation of the SNCA region using a complementary approach, under the hypothesis that conserved non-coding regions of the SNCA gene are comprised of transcriptional enhancers or silencers and thus modulate gene expression. This would mean that single nucleotide polymorphisms (SNPs) in these regions could influence the transcriptional pattern of the SNCA gene27.\n\n\nMaterials and methods\n\nUsing comparative genomics, we searched for highly conserved non-coding sequences between human and mouse and identified 37 evolutionary conserved non-coding genomic regions (ncECRs) within the SNCA gene that are conserved between human and mouse.\n\nWe utilized two complementary browsers (Vista browser (http://pipeline.lbl.gov/cgi-bin/gateway2) and ECR browser (http://ecrbrowser.dcode.org/) to generate a conservation profile by aligning the human SNCA gene with its mouse counterpart in a pair-wise fashion. We applied established selection parameters for our search with >100bp in length and >75% identity28,29. In addition to the 111.4kb SNCA gene region, we included a 44.5kb upstream and a 50kb downstream intergenic region to also capture surrounding regulatory elements.\n\nWe identified 37 ncECRs in the SNCA genomic region of 206kb on chromosome 4q21 (Chr.4: 90,961056-91,167082, UCSC Genome Browser on Human Mar. 2006 Assembly) by pair-wise comparison between human and mouse (Figure 1). Ten of these DNA sequences were located downstream of the SNCA gene, 17 were intronic between exon 4 and 5, which is 92kb in length, and five were upstream of the SNCA gene (Figure 1). None of the selected sequences overlapped with known expressed sequence tags (ESTs) or had an open reading frame of more than 20 amino acids in length, suggesting that these ncECRs are non-coding.\n\nPanel shows human-mouse pair-wise comparison of Human genome May 2004 and Mouse Sept. 2005. Pink marked peaks represent ncECRs, turquoise marked peak represent the untranslated region (UTR) of SNCA, blue marked peaks represent exons. D1-D10 are conserved regions downstream of SNCA. In1-In17 are intragenic conserved regions, and U1-U4-2/3 are upstream of SNCA. The black arrow on top shows the transcription orientation.\n\nTo test, if the ncECRs exhibit enhancer or silencer activity, we cloned all identified regions in specific reporter vectors and measured their luciferase activity after transfection into neuroblastoma cells. For our studies, we used the pGL3 luciferase reporter vectors (Promega, Cat. No. E1751, E1741, E1771, E1761) and the human neuroblastoma cell line SK-N-SH. NcECRs identified through the comparative analysis (Supplementary Table 1) were cloned upstream of a SV-40 promoter in the pGL3 promoter construct, transfected in SK-N-SH cells and assayed with the Dual-Luciferase® Reporter Assay System (Promega, Cat. No. E1910).\n\nSome of these regions were combined in one vector because of their close proximity to each other. Primers with specific restriction sites (KpnI, BglII or XhoI from New England Biolabs Inc.) were designed to amplify the conserved elements, and PCR products with specific restriction sites were directly cloned into the pGL3 promoter vector to ensure correct orientation of the genomic elements (Supplementary Table 1). All constructs were sequenced to ensure that no point mutations were introduced through the amplification and/or cloning process.\n\nFor transfection experiments, we used a 96-well format (Nunc, Cat. No. 167008). Cells were plated one day before transfection at a density of 3000–5000 cell/well to reach 90–95% confluency at the time of transfection, luciferase assays were performed 24hrs after transfection. SK-N-SH cells were maintained in Hyclone DMEM media (High Glucose, Fisher Scientific, Cat No. SH30081.02) with 10% Hyclone fetal bovine serum (Fisher Scientific, Cat No. SH30910.03) in 1× glutamine (Life Technologies, Cat. No. 25030-081) and 1× penicillin/streptomycin (Life Technologies, Cat. No. 15140-122). For SK-N-SH cells, we used 1:2 ratio of nucleic acid to transfection reagent (Lipofectamine® 2000 Transfection Reagent, Life Technologies, Cat No. 11668-019). For the luciferase assay, we used the Dual-Luciferase® Reporter (DLR™) Assay System (Promega, Cat. No. E1910) according to the manufacturer’s instructions in 96-well white plates, flat bottom (E&K Scientific, Cat. No. EK-25075). In this assay, activities of firefly and Renilla luciferases were measured sequentially in one sample. All assays were performed in quadruplicate and each experiment was repeated three times. Altogether, 12 data points were ascertained for each conserved region/construct.\n\nTo estimate the number of potential TFBSs and the number of interacting transcription factors (TFs) that could represent potential candidate proteins for our positive ncECRs, we used MatInspector in an in silico approach. We chose two elements for this bioinformatic analysis with MatInspector. The MatInspector software utilizes a large library of matrices for TFBSs to locate matching DNA sequences. The program assigns quality rating to matches and allows quality-based filtering and selection of matches. MatInspector can group similar or functionally related TFBSs into matrix families30.\n\nIn addition to the original human-mouse comparison, we added the sequences for dog and cow for comparisons. Only the TFBSs were considered that were present in all four species, in the same orientation, and similar distance to each other. We ran two analyses with 10 and 15 nucleotides distance, respectively. We accepted only models in which at least four TFs can bind in a concerted way. Each TFBS can potentially bind several TFs.\n\nWe also computationally tested all possible TFs for interactions with the SNCA promoter region, which were retrieved from the proprietary ElDorado database (Genomatix, Munich, Germany). In this database, promoters are defined and ranked by transcription start sites, corresponding known mRNA or EST sequences and by orthologous conservation.\n\n\nResults\n\nOverall, 12 of 37 conserved non-coding elements exhibited either an increase or reduction of the expression of the luciferase reporter gene (Figure 2 and Dataset 1). Three elements upstream of the SNCA gene (U3, U4-1, and U4-3) displayed a significant approximately 1.5 fold (p<0.009) increase in expression (Figure 2A). Of the intronic regions, three showed a 1.5 fold increase (I2, I6, I8) and two others showed a 2 and 2.5 fold increase in expression (p<0.002), I5 and I12, respectively (Figure 2B). Two elements downstream of the SNCA gene showed approximately 2 fold (D1 and D2) and 2.5 fold (D3) increase (p<0.0009) (Figure 2C). One element D6 downstream of SNCA had a reduced expression of the reporter gene of 0.35 fold (p<0.0009) of normal activity (Figure 2C, green) that was also confirmed after cloning the D6 element in a pGL3 control vector (Figure 2C, insert). The pGL3 control vector contains the SV-40 promoter and a SV-40 enhancer element. The D6 element reduced the expression of the pGL3 control construct by ~50%, confirming that this element represents a repressor. Between 4 and 12 replicates were performed per ncECR.\n\nPanels A–C show the luciferase assay results of ncECRs upstream (A), intragenic (B), and downstream (C) of the SNCA gene. The X-axis shows the ncECRs, the Y-axis shows the ratio of luciferase and renilla expression as percentage. Bas=pGL3 basic, Con=pGL3 control, prom=pGL3 promoter construct. All red or green box plot elements represent ncECRs that modulate expression significantly. The box plots show the median (horizontal line within box), the 25 and 75% tiles (horizontal borders of box), and the whiskers show the minimal and maximal values. Panel C, insert: Luciferase assay results of D6 element cloned into the pGL3 control vector construct.\n\nThese data provide experimental evidence that a significant proportion of the ncECRs show a regulatory function in the luciferase reporter assay.\n\nWe performed MatInspector (Genomatix) analysis30 on two elements (I12 and D6) with the highest fold change in the luciferase assay. In addition to the original human-mouse comparison to identify the ncECRs, we added the sequences from dog and cow. Only TFBSs that were present in all four species, in the same orientation, and similar distance to each other were considered. We ran two analyses with 10 and 15 nucleotides distance, respectively. We accepted only models in which at least four TFs can bind in a concerted way. Each TFBS can potentially bind several TFs. Interestingly, using this more restricted model, five factors showed an interaction with the SNCA promoter as well as with the ncECRs (Figure 3A). These factors were the Paired-like homeodomain transcription factor 3 (PITX3), the Homolog of Drosophila orthodenticle 2 (OTX2), the Nuclear receptor subfamily 3, group c, member 1 (NR3C1) or glucocorticoid receptor (GCCR), the Androgen receptor (AR), and the general transcription initiation factor TATA box-binding protein (TBP).\n\nA. Scheme of SNCA interaction with TFs that also potentially bind to two ncECRs within the SNCA gene. B. UCSC Genome browser custom track of PD associated SNPs (based on PD Gene metaanalysis), Rep1 allele and functional ECR regions on chromosome 4 (Human Genome Assembly Feb. 2009, GRCh37/hg19).\n\nIt is intriguing to note that by searching for TFs that bind to both the promoter and the functional ncECR, several DNA-binding proteins were found that are linked to dopaminergic regulation and susceptibility for nigrostriatal impairment. Two of these TFs (PITX3 and OTX2) implicated in determination of a dopaminergic phenotype in the substantia nigra emerged from this preliminary search31,32. PITX3 has shown to be regulated in a negative feedback circuit through the microRNA mi-133b to fine-tune maintenance of dopaminergic neurons33. In an association study, a SNP in the PITX3 promoter was reported to be associated with PD and might dysregulate expression of PITX334 suggesting that transcription factors play a critical role not only in the development and differentiation of dopaminergic neurons, but also for cell maintenance and survival of dopaminergic neurons.\n\nGCCR and AR belong to a class of nuclear receptors called activated class I steroid receptors. GCCR is a cytosolic ligand-activated transcription factor that regulates the expression of glucocorticoid-responsive genes. GCCR shows strong anti-inflammatory and immunosuppressive effects. Interestingly, impaired GCCR expression in a mouse model shows a dramatic increase in the vulnerability of the nigrostriatal dopaminergic neurons to a toxic insult of MPTP35.\n\nTaken together, this preliminary in silico screen resulted in very intriguing new candidates that might directly regulate SNCA expression and could play a role in the pathological processes that underlie PD.\n\n\nDiscussion\n\nA major focus in PD research has been on post-translational modification of α-syn. The alterations seen in PD that were linked to disease pathogenesis were nitrated α-syn and α-syn phosphorylated at serine 129 identified in Lewy bodies and Lewy neurites36,37, however, the gene transcription as a control point and its regulation in particular cell types or upon cellular signals has only been touched fairly recently in PD-relevant genes.\n\nOur results show that potential regulatory regions are not restricted to the promoter of the SNCA gene as discussed in the introduction, but are likely to be located also in other intronic and intergenic regions (Figure 3B). Comparing our results to similar screens, where conserved regions range from 8–45 elements38,41, we found a similar number of functional elements in our screen that show a high evolutionary conservation.\n\nNot only the promoter region of a gene drives the transcription/expression of a gene. Also other cis-acting genomic regions within a certain gene, up to several hundred kb away, can serve as enhancers, silencers, or modifiers to ensure the accurate temporal and spatial expression of a gene by recruiting transcription activating or silencing factors that bind to them38. There is ample precedence for this approach to analyze genomic regions of genes implicated in human disease. Mutations in those conserved elements were found to cause human genetic syndromes, for example SALL1/Townes-Brocks syndrome39 or SHH/preaxial polydactyly40. Other groups have investigated the non-coding regulatory elements within disease genes such as RET (Ret proto-oncogene) and MECP2 (Methyl-CpG binding protein 2) and found multiple regulatory enhancer and silencer elements38,41.\n\nSpecific TFs seem to be directly involved in neurodegeneration and models of PD. TFs have been shown to be critical regulators for the development, maintenance and survival of dopaminergic neuronal populations42,43. E.g. forkhead transcription factor (Foxa2) is responsible for early development of endoderm and midline structures. Foxa2 is specifically expressed in postmitotic dopaminergic neurons. Genetically engineered mice that are null for Foxa2 are not viable, whereas heterozygotes for Foxa2 develop major motor abnormalities starting at 18 months with an asymmetric posture, rigidity, and bradykinesia44.\n\n\nConclusion\n\nThis screen of evolutionary conserved genomic elements in the SNCA locus showed a number of functionally elements that in an in vitro assay modulated the expression of a reporter gene. Furthermore, we identified very intriguing new candidate transcription factors that could directly regulate SNCA expression and could, if binding is altered by genetic variants, play a role in the pathological processes that underlie PD. This is the first step to systematically analyze the SNCA locus to understand its transcriptional regulation in more detail. Further studies are needed in neuronal tissues (e.g. dopaminergic neurons derived from patient-specific induced pluripotent stem cells) to confirm these findings and expand the analysis to identify SNCA-regulating transcription factors. By defining the transcription factors that regulate expression and potentially overexpression of α-synuclein that can lead to neurodegeneration, we will be able to identify targets for novel therapeutic approaches for α-synucleinopathies including Parkinson’s disease.\n\n\nData availability\n\nF1000Research: Dataset 1. Combined normalized raw datasets of Luciferase assays on SNCA conserved elements, http://dx.doi.org/10.5256/f1000research.3281.d3745245", "appendix": "Author contributions\n\n\n\nBS conceived the study and designed the experiments, and drafted the manuscript. LS carried out the experiments. DT analyzed data. MW carried out in silico analysis for transcription factors. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by a pilot grant of NIEHS-CCPDER 1U54ES012077 to B. Schüle (PI: J.W. Langston), and by the Parkinson’s Unity Walk.\n\n\nAcknowledgements\n\nPart of the content of this manuscript has been presented as a poster at the Annual Meeting of The American Society of Human Genetics 2007:\n\nSchüle, B., Sterling, L., Langston J.W.: Characterization of cis-regulatory elements in the alpha-synuclein gene; (Abstract, http://www.ashg.org/genetics/ashg07s/f21298.htm). Presented at the Annual Meeting of the American Society of Human Genetics, October 23–27 in San Diego, CA, USA\n\n\nSupplementary Table\n\n\n\n\nReferences\n\nCookson MR: alpha-Synuclein and neuronal cell death. Mol Neurodegener. 2009; 4: 9. 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Data Source" }	[ { "id": "6592", "date": "06 Nov 2014", "name": "Ornit Chiba-Falek", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper by Dr. Schüle’s team describes on the identification of evolutionary conserved non-coding regions (ncECRs) in the α-synuclein (SNCA) gene and their assessment as candidate regulatory elements. The work coupled in silico and cell-based studies. By using a comparative genomic screen between human and mouse the authors identify 32 ncECRs, out of which 11 regions exert an effect on expression level using a luciferase reporter assay approach. Their findings add on previous reports in the field that have shown, using both luciferase reporter system and human brain tissues, that the SNCA gene contains cis-regulatory sites across the 3’ and the 5’ LD blocks that regulate its expression levels.The study was well designed and thoroughly executed, the results are of interest to the scientific community of PD-genetics, and provide seeds for follow up studies. The paper is nicely written, logically flows and summarizes the literature in the field. However, the authors should make major revisions according to the following comments:There is some inconsistency regarding the number of the ncECRs identified in the initial screen between the different sections of the article (32, 34, 37). Please make the corrections where needed. Additional necessary control for the Luciferase experiments is a pGL-(SV40) promoter vector harboring an insert of a scrambled sequence that its size range mimics the average insert size of the tested ECRs. This is required to control for the ‘spacer’ effect of ECR lengths. What method was used for the statistical analysis? It is also not clear in the text whether all significant changes were calculated in comparison to the SV-40 promoter-only vector. That should be described in details in the method section. To demonstrate the important implication of this study the authors are recommended to follow up on an event as an example. That is to say, to evaluate the effect of a genetic variation, a PD-associated SNP, on the regulatory function of the corresponding ECR using the luciferase system established in this work. Figure 3 demonstrates overlap between PD associated SNPs and ncECR, connecting these dots will be of high significance. Supp Table: there is a typo in the coordinates of D2. In the footnote include the human genome assembly of the coordinates. Figure 2A X-axis: modify title to ‘upstream….’ Omit Figure 3A. Instead include a new panel to figure 3B that indicates the position of the putative binding sites of these TFs within SNCA locus. The identification of Transcription Factor Binding Sites (TFBS) is an important step required in order to evaluate the transcriptional regulation network of the SNCA gene. To this end, the computational prediction of TFBS is a classic approach that gives preliminary data but should be interpreted with caution. Integration of the classic approach with new models described in Mathelier & Wasserman (2013) is highly recommended. The relation between TF motifs and in vivo binding sites is far from simple. The analysis lacks of information about the context of the identified sequences. TF are highly context-specific, and the same TF typically binds to different genomic binding sites in different conditions. Obtaining information about the context could be helpful in better understanding the possible involvement of the predicted sites as TFBS. While this is beyond the scope of this study, this topic should be thoroughly discussed in the discussion section.", "responses": [ { "c_id": "1097", "date": "26 Nov 2014", "name": "Birgitt Schuele", "role": "Author Response", "response": "We very much appreciate the careful review and excellent comments, suggestions and future directions of the reviewers. We hope to have addressed all of the comments to the reviewers’ satisfaction. There is some inconsistency regarding the number of the ncECRs identified in the initial screen between the different sections of the article (32, 34, 37). Please make the corrections where needed.Thanks so much for the comment. We made changes to reflect the correct number of 34 ncECRs. We combined counts for ncECRs that were located very closely in the luciferase assay to one ncECR therefore different numbers appeared in the text. That has been addressed. Additional necessary control for the Luciferase experiments is a pGL-(SV40) promoter vector harboring an insert of a scrambled sequence that its size range mimics the average insert size of the tested ECRs. This is required to control for the ‘spacer’ effect of ECR lengths.We have included in our analysis three controls: 1. The pGL3-Basic Vector which lacks eukaryotic promoter and enhancer sequences should not show any transcription activity. 2. The pGL3-Enhancer Vector contains an SV40 enhancer located downstream of luc+ and the poly(A) signal and is showing transcription a very high levels (enhancer element is 246bp in length). 3) pGL3-Promoter Vector contains an SV40 promoter upstream of the luciferase gene (promoter is 202bp in length). Even though we have not directly included a control with scrambled sequence, we think that the ncECR elements that do not change transcription of luc+ provide enough evidence that the experimental system is valid. Of a total of 34 in silico determined elements, only 12 show an effect of transcriptional regulation. 22 elements did not change expression compared to pGL3-Promoter Vector. What method was used for the statistical analysis? It is also not clear in the text whether all significant changes were calculated in comparison to the SV-40 promoter-only vector. That should be described in details in the method section. A description of the analysis of luciferase assays was lacking and has now been added as a paragraph at the end of Method section Cloning and luciferase assays and reads as follows:“Statistical analysis: Differences among means were analyzed using two-samples student’s t-test. For differences in transcriptional activation of the luc+ gene, ncECRs were tested in quadruplicates in three independent experiments. Differences were considered statistically significant at p<0.05.”To demonstrate the important implication of this study the authors are recommended to follow up on an event as an example. That is to say, to evaluate the effect of a genetic variation, a PD-associated SNP, on the regulatory function of the corresponding ECR using the luciferase system established in this work. Figure 3 demonstrates overlap between PD associated SNPs and ncECR, connecting these dots will be of high significance.This is an excellent suggestion and will definitely be conquered in future work with this system as this is the basis for the understanding of transcriptional regulation of the SNCA locus for potential translational applications. The presented study was intended to understand the basic changes in transcriptional regulation within the SNCA locus. Supp Table: there is a typo in the coordinates of D2. In the footnote include the human genome assembly of the coordinates.We corrected the coordinates for D2 which was a duplicate of D1 with the correct genomic location chr4:90844830+90845413 and added in the header the corresponding Human Genome assembly NCBI36/hg18 (March 2006).Figure 2A X-axis: modify title to ‘upstream….’Correction has been made. It reads now in Figure 2A “Upstream SNCA conserved elements”. We also changed for consistency Figure 2B to “Intronic SNCA conserved elements” and capitalized Figure 2C “Downstream SNCA conserved elements”.Omit Figure 3A. Instead include a new panel to figure 3B that indicates the position of the putative binding sites of these TFs within SNCA locus.We have modified Figure 3 according to the MatInspector network view with respective changes in the legend. We also included which genomic sequences have been analyzed in the text. Since this is a preliminary in silico analysis, we feel that the overview is sufficient and has to be validated in functional studies. As pointed out below by the reviewer, these analyses have to be taken with care and a grain of salt. The identification of Transcription Factor Binding Sites (TFBS) is an important step required in order to evaluate the transcriptional regulation network of the SNCA gene. To this end, the computational prediction of TFBS is a classic approach that gives preliminary data but should be interpreted with caution. Integration of the classic approach with new models described in Mathelier & Wasserman (2013) is highly recommended. The relation between TF motifs and in vivo binding sites is far from simple. The analysis lacks of information about the context of the identified sequences. TF are highly context-specific, and the same TF typically binds to different genomic binding sites in different conditions. Obtaining information about the context could be helpful in better understanding the possible involvement of the predicted sites as TFBS. While this is beyond the scope of this study, this topic should be thoroughly discussed in the discussion section.Thank you very much for this suggestion. Indeed, further studies are necessary to provide experimental evidence for the binding of predicted transcription factors. The analysis provided in this article was only a first step to model potential transcription factor binding sites and should stimulate further studies.The reference Mathelier and Wasserman has been now included in the Discussion of the manuscript and reads as follows: “Computationally determining transcription factor binding sites is a challenging process and multiple prediction algorithms have been developed over the last decade (Cartharius 2005, Wu 2009, Mathelier 2013). Therefore our preliminary data should solely open the discussion and drive novel hypotheses for potential transcription factors that regulate transcription of the SNCA locus.”" } ] }, { "id": "6590", "date": "10 Nov 2014", "name": "Jinglan Liu", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article by Sterling et al. has described the identification and functional analysis of evolutionally conserved non-coding elements that might be involved in the transcriptional regulation of the gene SNCA, mutations in which were associated with Parkinson’s disease. This is a very interesting, proof-of-concept article, with an attempt to provide pathogenic insight from the point of view of regulatory genomics for a complex human disease. I endorse the indexing of this manuscript. It is now well recognized that ~98% of human genome do not code for proteins. Comparative genomics studies revealed that the majority of evolutionally conserved regions consist of non-coding elements that that might be involved in regulating gene expression. Genome-wide association studies (GWAS) have showed that the majority (~93%) of SNPs contributing to human diseases or susceptibility lie outside protein-coding regions, and there are many non-coding SNPs have been demonstrated to be associated with common diseases and traits. By identifying functionally significant non-coding elements for SNCA, Sterling et al.’s workcould lend a new perspective to study the genetic architecture of Parkinson’s disease, and promote further investigations on the pathogenic impact of non-coding elements and their regulatory networks on the clinical courses of Parkinson’s disease.", "responses": [] } ]	1	https://f1000research.com/articles/3-259
https://f1000research.com/articles/3-265/v1	04 Nov 14	{ "type": "Research Note", "title": "Correlating the ability of VP24 protein from Ebola and Marburg viruses to bind human karyopherin to their immune suppression mechanism and pathogenicity using computational methods", "authors": [ "Sandeep Chakraborty", "Basuthkar J. Rao", "Bjarni Asgeirsson", "Abhaya M. Dandekar", "Basuthkar J. Rao", "Bjarni Asgeirsson", "Abhaya M. Dandekar" ], "abstract": "Immune response suppression is crucial for viral invasion. The protein VP24 is pivotal in achieving this in Ebola, although interestingly the mechanism of immune suppression is different in the closely related Marburg virus. Here, we illustrate that a possible molecular basis for this diffrence emanates from two alpha helical structures (α5 and α6) in VP24 involved in binding human karyopherin (KPNA) (PDBid:4U2X), wherein the Ebola and Marburg viruses have distinctly different charged properties in α5. α6 is absent in Marburg, and has a different hydrophobic moment in the Reston Ebola (REBOV) species, which is surprisingly non-pathogenic in humans. Based on the hypothesis that REBOV is not immunosuppressive, which is in turn is due to its inability to bind KPNA, we show by docking KPNA to the REBOV VP24 that the single amino acid substitution R140S is responsible for this difference between REBOV and Zaire Ebola strains. Such a scenario of getting a virulent REBOV through a single mutation is particularly worrisome, since the REBOV, once found only in monkeys, has been recently detected in pigs. We also reiterate the potential of using these helices as potential epitopes for generating protective antibodies against Ebola.", "keywords": [ "Viruses from the family Filoviridae are negative-stranded RNA viruses having a filamentous shape1. The first member of this family (Marburg) was discovered in 19672", "while the Ebola virus was first discovered in 19763. Public attention has been drawn to this rare", "but deadly disease4 ever since the current outbreak in West African countries threatened to rapidly deteriorate into a full blown epidemic5", "6. Both these viruses cause haemorrhagic fever by quickly suppressing innate antiviral immune responses7. However", "quite surprisingly", "the Reston Ebola (REBOV) strain", "first identified in monkeys and imported into the United States in Reston from the Philippines8", "is non-pathogenic in humans9", "10." ], "content": "Introduction\n\nViruses from the family Filoviridae are negative-stranded RNA viruses having a filamentous shape1. The first member of this family (Marburg) was discovered in 19672, while the Ebola virus was first discovered in 19763. Public attention has been drawn to this rare, but deadly disease4 ever since the current outbreak in West African countries threatened to rapidly deteriorate into a full blown epidemic5,6. Both these viruses cause haemorrhagic fever by quickly suppressing innate antiviral immune responses7. However, quite surprisingly, the Reston Ebola (REBOV) strain, first identified in monkeys and imported into the United States in Reston from the Philippines8, is non-pathogenic in humans9,10.\n\nPreviously, we have characterized α helical (AH) structures in Ebola proteins using PAGAL11, and demonstrated that the AHs with characteristically unique feature values are involved in critical interactions with the host proteins12. We show that the AH from Ebola virus membrane fusion subunit GP213, which is disrupted by a neutralizing antibody derived from a human survivor of the 1995 Kikwit outbreak14, has a very large hydrophobic moment compared to other AHs in Ebola proteins12. Similarly, another AH with the highest proportion of negatively charged residues is the binding site of the human karyopherin (KPNA) to the Zaire Ebola (ZEBOV) virus VP24 (ezVP24) protein15.\n\nIn spite of sharing a common ancestry16, Marburg and Ebola have different antigenicity of the virion glycoprotein17. Furthermore, the mechanism of immunosuppression is different in these viruses18. These differences are probably the reason for the lesser mortality observed in Marburg outbreaks. In Ebola, the crucial role of host immune system evasion is accomplished by two proteins: VP35 and VP2419. Ebola VP24 inhibits interferon (IFN) signaling by hindering the nuclear accumulation of tyrosine phosphorylated STAT1 by binding KPNA20,21. In contrast, the Marburg virus abrogates the host immune response by inhibiting IFN induced tyrosine phosphorylation of STAT1 and STAT218 via the moonlighting matrix protein, VP4022. Specifically, ezVP24 binds KPNA via two AHs (α5 and α6)15. In Marburg VP24 (mVP24), α5 has distinctively different properties (not easily identified by a sequence or structural alignment), while α6 is just a small turn23. This rationalizes why mVP24 is not immunosuppressive.\n\nWe investigated these AHs in VP24 from the REBOV strain (erVP24). While α5 in erVP24 was similar to that in ezVP24, α6 in erVP24 was found to have different properties, caused by the presence of a serine in the place of arginine (S140R). We modelled the apo erVP24 (PDBid:4D9OA) using the ezVP24 in complex with KPNA as a template (PDBid:4U2X) by SWISS-MODEL24, and then docked KPNA to this structure using DOCLASP25. The docked structure helped in visualizing the ability of Arg140 in ezVP24 to make the correct electrostatic interaction with two glutamic acids, one of them residing on α5 in VP24, and the other in KPNA. The effect of single mutations in modulating virulence has been well established26–28. However, our methodology provides a more rational way of finding such critical residues. The possibility of a REBOV mutant gaining immunosuppressive capabilities is particularly disconcerting ever since the isolation of the REBOV strains from pigs29–31. We also highlight the possibility of using α5 and α6 from VP24 as epitopes for generating antibodies32, or designing compounds and peptides to inhibit protein-protein interaction33.\n\n\nMaterials and methods\n\nAHs in proteins were identified using DSSP34. These AHs were then analyzed using PAGAL11. Briefly, the Edmundson wheel is computed by considering a wheel with centre (0,0), radius 5, first residue coordinate (0,5) and advancing each subsequent residue by 100 degrees on the circle, as 3.6 turns of the AH makes one full circle. We compute the hydrophobic moment by connecting the center to the coordinate of the residue and give it a magnitude obtained from the hydrophobic scale (in our case, this scale is obtained from35). These vectors are then added to obtain the final hydrophobic moment. The color coding for the Edmundson wheel is as follows: all hydrophobic residues are colored red, while hydrophilic residues are colored in blue: dark blue for positively charged residues, medium blue for negatively charged residues and light blue for amides.\n\nThe protein structures used in the current work are all identified using the PDBid, and are available at www.rcsb.org. We used the SWISS-MODEL program to model the erVP24 (PDBid:4D9OA) structure using the ezVP24 (PDBid:4U2XA) in complex with KPNA as template. See 4D9OA4U2XA.pdb in Dataset 1. Note the residue numbering is not conserved by SWISS-MODEL. For example, Glu113 in PDBid:4D9OA corresponds to Glu97 in PDBid:4D9OA4U2XA. We used DOCLASP25 to dock KPNA to the modelled structure of erVP24 (See Pymol script ‘dockingKPNAtoRestonVP24.p1m’ in Dataset 1). ‘4U2XA.4U2XD.maxdist.out.sort’ in Dataset 1 lists the closest atoms of the residues of VP24 (PDBid:4U2XA) that make contact with human karyopherin (PDBid:4U2XD), sorted based on distances.\n\nAll protein structures were rendered by PyMol (http://www.pymol.org/). The sequence alignment was done using ClustalW36. The alignment images were generated using SeaView37. Protein structures have been superimposed using MUSTANG38.\n\n\nResults and discussion\n\nezVP24 has a 39.6% identity (73.8% similar) with mVP24 (Figure 1a), and there is significant structural homology among VP24 proteins from different strains of Ebola and Marburg (Figure 1b). Yet, the mechanism of immune response suppression is different in these viruses from the Filoviridae family18. ‘Reasons why Marburg virus VP24 is not immunosuppressive remain elusive’23. Therefore, we sought to investigate the differences in residues involved in binding KPNA in the ezVP24 and mVP24.\n\n(a) EbZaire: Zaire Ebola, EBSudan: Sudan Ebola, EBReston: Reston Ebola, Mar-Musoke: Marburg Musoke. Multiple sequence alignment was done using ClustalW. (b) Structural alignment of PDBid:4M0QA (Ebola Zaire Apo, in red), PDBid:4U2XA (Ebola Zaire complexed, in green), PDBid:4D9OA (Ebola Reston Apo, in blue), PDBid:3VNEA (Ebola Sudan Apo, in yellow) and PDBid:4OR8A (Marburg Musoke Apo, in orange). Structural alignment was done using MUSTANG38. (c) Helices involved in binding human karyopherin (α5 and α6 in magenta). Note, that the α5 is not a helix in Marburg VP24 (PDBid,4OR8A, in orange), but just a small turn.\n\nezVP24 binds KPNA via two AHs (α5 and α6), residues on loops and a Lys on a β-sheet (Table 1). In mVP24, α5 has different properties (Figure 2a,b and Table 2), while α6 is just a small turn (Figure 1c). These differences in the properties of AHs involved in binding KPNA in eVP24 to those in mVP24 strongly indicates that mVP24 is not immunosuppressive, as is widely accepted18 (at least, it does not have the same mechanism).\n\nOne or more atoms from these residues are within 4 Å of residues from human karyopherin.\n\nThe color coding for the Edmundson wheel is as follows: all hydrophobic residues are colored red, while hydrophilic residues are colored in blue: dark blue for positively charged residues, medium blue for negatively charged residues and light blue for amides. (a) Apo ezVP24 (PDBid:4M0QA). (b) Apo mVP24 (PDBid:3VNEA). It can be seen that mVP24 has two positively charged residues in the AH, unlike eZVP24. (c) ezVP24 (PDBid:4U2XA) in complex with human karyopherin (PDBid:4U2XD). Note, that Glu113 and Pro114 are now part of the AH, in contrast to the apo AH in (a). (d) Apo erVP24 (PDBid:4D9OA).\n\nIt can be seen that the Marburg VP24 (mVP24) protein has a distinctly different charge residue composition in the helix. This strongly indicates that mVP24 might not bind human karyopherin, which is the mechanism of immunosuppression by the Ebola VP24 proteins. HM: Hydrophobic moment, RPNR: Ratio of the positive to the negative residues, Len: length of the helix, NCH: number of charged residues.\n\nThe REBOV strain ‘does not represent an immediate public health menace on the scale of the African Ebola virus’9, possibly due to the generation of antibodies against this strain39. Also, gene expression of infected cells that ZEBOV and Marburg viruses showed fewer activated IFN-inducible genes relative to REBOV40. Thus, most likely, the REBOV strain does not have the same immunosuppressive capabilities of the ZEBOV or Sudan strain. While α5 of erVP24 has properties similar to ezVP24 (Figure 2c), α6 in REBOV VP24 (erVP24) is clearly different (hydrophobic moment, residue composition) in REBOV (Figure 3). For example, Arg140 in ezVP24 is replaced with Ser140 in erVP24.\n\n(a) apo ezVP24 (PDBid:4M0QA). (b) ezVP24 in complex with humans karyopherin (PDBid:4U2X). Note, that the AH is extended by two residues (E143 and Q144) as compared to the apo protein. However, the hydrophobic moment remains the same. (c) α6 of esVP24 (PDBid:3VNEA). (d) α6 of erVP24 (PDBid:3VNEA). It can be seen REBOV VP24 has a different hydrophobic moment than the other, since Ser140 is place of Arg140.\n\nIn order to better visualize this difference, and to quantify it, we docked KPNA to erVP24. First, we modelled the apo erVP24 (PDBid:4D9OA) using the ezVP24 complexed with KPNA (PDBid:4U2X) using SWISS-MODEL24. Subsequently, KPNA was docked to this protein using DOCLASP25.\n\nFigure 4 shows the ezVP24 and erVP24 docked to KPNA. In ezVP2, KPNA binding is primarily facilitated by electrostatic attraction between the negatively charged Asp124 in α5 and Lys481 in KPNA (at 3.9 Å)12, and a hydrogen bond between Arg140 (α6) and Glu475 of KPNA (among other hydrogen bonds, Table 3). Also, the ezVP24 itself is stabilized by an electrostatic bond between the negatively charged Glu113/OE1 (α5) and the positively charged Arg140/NH1 (α6) at 3.4 Å. Note, that this pair is at distance of 12.8 Å in the apo ezVP24 (PDBid:4M0QA). This 8 Å conformational change in these AHs emphasizes the role of plasiticity in binding KPNA. In contrast, in the erVP24, the distance between Glu113/OE1 and Ser140/OG changes from 14 Å in the apo enzyme to 6.2 Å in the docked model. Also, Ser140/OG atom is not positively charged unlike Arg140/NH1. Further, the possibility of Ser140/OG making a hydrogen bond with Glu475 of KPNA is remote, since they are 6.7 Å apart. Thus, we conclude that the mutation R140S is likely to be responsible for the non-pathogenic nature of REBOV, since this mutation renders erVP24 incapable of binding KPNA.\n\nThe erVP24 was modelled using SWISS-MODEL24 using ezVP24 structure complexed with KPNA (PDBid:4U2XA) (See 4D9OA4U2XA.pdb in Dataset 1). The docking was done using DOCLASP25, which superimposes the proteins as well. (a) Superimposition of modelled erVP24 and ezVP24, with bound KPNA. (b) Electrostatic attraction between the negatively charged Glu113/OE1 (α5) and the positively charged Arg140/NH1 (α6) at 3.4 Å, and a hydrogen bond between Arg140 (α6) and Glu475 of KPNA stabilizes the binding. (c) Ser140 replaces Arg140 in erVP24, and fails to make any of the above interactions.\n\nThe complete sorted list can be found in ‘4U2XA.4U2XD.maxdist.out.sort’ in Dataset 1. Note, that there is a hydrogen bond between Arg140/NH2 and Glu475/O.\n\nIt is interesting to note that the apo α5 (PDBid:4M0QA) is extended by two residues towards the N-terminal (Figure 2c, Glu113 and Pro114) in the ezVP24 complex with KPNA (PDBid:4U2XA). Notably, Pro and Glu are the two most disorder promoting residues41. The peptide stretch preceding Glu113 in the Sudan Ebola VP24 (PDBid:3VNEA) is also disordered, and residues in that stretch are unassigned in the crystal structure (Figure 1a). Quite interestingly, the α6 (Figure 3a) is also extended by two residues (towards the C-terminal) in the ezVP24 complex (Figure 3d). As mentioned earlier, this stretch is not a helix in mVP24. In the apo Sudan Ebola VP24, α6 (Figure 3c) is similar to the ezVP24 complex (Figure 3b), and is already extended. This is probably due to the fact that Glu is replaced by Asp, which is not disorder generating. Also, the hydrophobic moment of all three AHs have (almost) the same direction and magnitude (Figure 3a–c). These observations emphasizes the role of intrinsically disordered regions in viral functionality42,43.\n\n\nConclusions\n\nThe ability of a single mutation to significantly alter the immunosuppressive properties of the Ebola proteins is well established26,27,44. Sequence based methods (whole genome profiling) are typically used to identify these critical mutations26. Structural studies provide an alternate, and possibly more rational, method to identify such mutations. For example, while double (and not single) mutations are required in VP35 to inhibit protein kinase R activation, it is difficult to rationalize this based on sequence data only28. In the current work, we build on previous work that has characterized AH structures in the Ebola proteome to rationalize the lack of immunosuppressive properties in the mVP24. ezVP24 binds to KNPA via two AHs (α5 and α5), loops and a residue on a β-sheet. We attribute the lack of immunosuppressive properties of mVP24 to its inability to bind KPNA, which emanates from different characteristics of α5 of mVP24 compared to ezVP24. Subsequently, we demonstrate that a single mutation in α6 in the erVP24 might endow it with immunosuppressive properties. We corroborate this conclusion by modelling the apo structure of the erVP24 based on the structure of ezVP24 in complex with KPNA using SWISS-MODEL24, and docking KPNA to the modelled structure using DOCLASP25. The REBOV strain, first identified in monkeys and imported into the United States from the Philippines8, has never caused disease in humans9,10. However, the isolation of the REBOV strains from pigs in Philippines29,30, and recently in China31, highlights the significance of finding preventive therapies in the probable scenario a mutant REBOV for VP24 with immunosuppressive capabilities gets transferred to human handlers. Such a difference does not exist in the VP35 protein, where REBOV VP35 has been used as a model to show how they could silence and sequester double-stranded RNA, which are key events in immunosuppression45. We also reiterate the potential of using these AHs from VP24 as epitopes46,47 for generating antibodies32,48,49, or innovating drugs to inhibit protein-protein interaction33,50–54. The presence of two intrinsically disordered residues proximal to these AHs in the apo structure that gain a AH structure upon binding should encourage antibody search to use both apo and complexed AHs. It is certainly worth investigating whether supplementing ZMapp, a cocktail of three antibodies has shown reversion of advanced Ebola symptoms in non-human primates55, with more antibodies would prove more effective.\n\n\nData availability\n\nF1000Research: Dataset 1. Docking human karyopherin to the Reston Ebola VP24 using Zaire Ebola VP24 as template using DOCLASP, 10.5256/f1000research.5666.d3806956", "appendix": "Author contributions\n\n\n\nSC wrote the computer programs. All authors analyzed the data, and contributed equally to the writing and subsequent refinement of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nAMD wishes to acknowledge grant support from the California Department of Food and Agriculture PD/GWSS Board. BJ acknowledges financial support from Tata Institute of Fundamental Research (Department of Atomic Energy). Additionally, BJR is thankful to the Department of Science and Technology for the JC Bose Award Grant. BA acknowledges financial support from the Science Institute of the University of Iceland.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nDolnik O, Kolesnikova L, Becker S: Filoviruses: Interactions with the host cell. Cell Mol Life Sci. 2008; 65(5): 756–776. PubMed Abstract \| Publisher Full Text\n\nKissling RE, Robinson RQ, Murphy FA, et al.: Agent of disease contracted from green monkeys. Science. 1968; 160(3830): 888–890. PubMed Abstract \| Publisher Full Text\n\nPattyn S, van der Groen G, Courteille G, et al.: Isolation of Marburg-like virus from a case of haemorrhagic fever in Zaire. Lancet. 1977; 1(8011): 573–574. PubMed Abstract \| Publisher Full Text\n\nColebunders R, Borchert M: Ebola haemorrhagic fever--a review. J Infect. 2000; 40(1): 16–20. 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PubMed Abstract \| Publisher Full Text\n\nWells JA, McClendon CL: Reaching for high-hanging fruit in drug discovery at protein-protein interfaces. Nature. 2007; 450(7172): 1001–1009. PubMed Abstract \| Publisher Full Text\n\nChapman RN, Dimartino G, Arora PS: A highly stable short alpha-helix constrained by a main-chain hydrogen-bond surrogate. J Am Chem Soc. 2004; 126(39): 12252–12253. PubMed Abstract \| Publisher Full Text\n\nBird GH, Madani N, Perry AF, et al.: Hydrocarbon double-stapling remedies the proteolytic instability of a lengthy peptide therapeutic. Proc Natl Acad Sci U S A. 2010; 107(32): 14093–14098. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBird GH, Boyapalle S, Wong T, et al.: Mucosal delivery of a double-stapled RSV peptide prevents nasopulmonary infection. J Clin Invest. 2014; 124(5): 2113–24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarrison RS, Shepherd NE, Hoang HN, et al.: Downsizing human, bacterial, and viral proteins to short water-stable alpha helices that maintain biological potency. Proc Natl Acad Sci U S A. 2010; 107(26): 11686–11691. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQiu X, Wong G, Audet J, et al.: Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp. Nature. 2014; 514(7520): 47–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChakraborty S, Rao B, Asgeirsson B, et al.: Dataset 1 in: Correlating the ability of VP24 protein from Ebola and Marburg viruses to bind human karyopherin to their immune suppression mechanism and pathogenicity using computational methods. F1000Research. 2014. Data Source" }	[ { "id": "6641", "date": "24 Nov 2014", "name": "Michael McIntosh", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article presents an interesting in silico observation to possibly explain observed differences in pathogenesis and suppression of host immune antiviral type 1 interferon (INF) responses emanating from structural differences in VP24 proteins of various Ebola virus (EBOV) species and Marburg virus. For context, host antiviral INF signaling is known to induce nuclear transport of tyrosine-phosphorylated signal transducer and activator of transcription 1 (STAT1) as an early stage in a signaling cascade that activates expression of host genes involved in antiviral mechanisms. A subset of the host Karyopherin alpha (KPNA) family are involved in the nuclear transport of activated STAT1, and EBOV VP24 protein has been shown by others (Xu et al., 2014) to bind KPNA thus interfering with this nuclear transport and the progression of host innate and adaptive immune responses to EBOV infection. Marburg virus is noted to interfere with host antiviral INF responses differently via direct inhibition of phosphorylation/activation of STAT1 and STAT2. In this article, in addition to gross charge and structural differences in two alpha helices (a5 and a6) of VP24 between EBOV and Marburg viruses, possibly explaining the different mechanisms of INF response suppression, the authors hypothesis that a single substitution R140S in VP24 between the pathogenic Zaire ebolavirus (ZEBOV) and non-pathogenic Reston ebolavirus (REBOV) alters charged properties of the a5 alpha helix leading to a lack of binding to human KPNA by REBOV VP24. This substitution in REBOV VP24 is hypothesized to be responsible for the lack of REBOV pathogenesis in humans. The authors further express concern regarding the potential for a single amino acid substitution in REBOV, previously observed in domestic swine, to perhaps lead to a more pathogenic virus in the future.Article Content:The study employs computational modeling of the primary VP24 amino acid sequences of different EBOV species and Marburg virus onto the previously resolved crystal structure of ZEBOV VP24 bound to KPNA5 (Xu et al., 2014). The direct comparisons between potential binding sites of KPNA and VP24 from different species of EBOV are intriguing but the study unfortunately lacks experimental verification either through in vitro binding or functional studies In addition there are concerns regarding the accuracy of theoretical modeling of primary VP24 sequences from various EBOV species to the known crystal structure of ZEBOV VP24 and KPNA5 peptides. Without experimental verification it is not possible to draw the conclusion that the R140S substitution present in REBOV affects binding to KPNA or that it is responsible for the absence of pathogenicity in humans. One approach not tried is modeling of a KPNA5 homolog from non-human primates as REBOV is known to still be pathogenic in non-human primates. In concept, it seems unlikely that a single mutation could be wholly responsible for the observed differences in pathogenicity between REBOV and other EBOV species. Various mechanisms not involving VP24 including EBOV glycoprotein and VP35-mediated mechanisms of immune suppression as well as a potential host genetic differences are likely to have critical influences on EBOV pathogenesis beyond the specific mechanism of VP24-mediated suppression of activated STAT1 nuclear localization and expression of INF triggered host antiviral mechanisms.Of minor importance, invasion should be replaced with pathogenesis in the first sentence of the abstract and minor typographical errors should be corrected.", "responses": [ { "c_id": "1108", "date": "01 Dec 2014", "name": "Sandeep Chakraborty", "role": "Author Response", "response": "Dear Dr McIntosh,'We would like to thank you for taking the time to review this paper, and for your insightful comments. While our method is computational, and there is no easy way to get around that fact for us with respect to Ebola, we do believe that dissemination of such information can provide direction in the effort to understand, and finally abrogate, the mechanism of pathogenesis of the Ebola virus. Recently, we have used the PAGAL 1 software to design anti-microbial peptides that work against plant pathogens 2.The logical thread of our hypothesis in this manuscript follows the inability of the VP24 from Marburg to bind KPNA owing to the difference in two helices (analzyed using PAGAL) that bind KPNA in the Zaire Ebola virus. We believe this point is irrefutable. A small difference in one of the helices (alpha6) in theVP24 from Reston Ebola virus results in two computationally arrived differences.Different hydrophobic moment in the Edmundson wheel (Fig3) (on a known structure, so confirmed). This difference is also visible in a multiple sequence alignment of the protein from different species. Different charged interactions of the residues in KPNA and VP24, after docking (on a modelled structure, possible inaccuracies).These differences might not have drawn attention, if Reston Ebola was not known to be non-pathogenic to humans. We have taken care at each point to clearly indicate that this is a possibility, and not a foregone conclusion. In fact, studying the ‘Reston-pathogenicity puzzle’ using deuterium exchange mass spectrometry (DEMS) methods, Zhang et. al. (2012) have identified putative sites which includes a ‘cluster of Reston-specific residues in VP24 is L136, R139 and S140’ 3. It is possible that these differences would not lead to loss of binding when such experiments are finally done, and we would have to revise our hypothesis (which theF1000Research model allows us to do). We emphasize on the role of computational methods to make intelligent and informed decisions, enabling biologist to design experiments, and minimizing human effort and cost - something that has been sorely missing in the Ebola effort.In this context, and also in response to your comment on the unlikelihood of a single mutation resulting in pathogenicity, we would like to cite recent work that identifies two mutations (one in VP24 and the other in the nucleoprotein) resulting in the acquisition of high virulence in mice 4. The VP24 mutation is Thr50,and lies on a beta-sheet, and its importance in the structure has not been completely understood to date, although this residue is another putative site in the DEMS study 3. Our group, that has focused on the importance of alpha-helices, but not beta-sheets 5, is trying to rationalize the overwhelming significance ofthis mutation.We also appreciate your idea of using KPNA from a non-human primate. However, only mice and rats have solved KPNAs. We have now included data on docking of a mouse KPNA to the Reston VP24 after conducting a similar analysis, and found no difference in their interactions (Fig. 5). Interestingly, we havealso come across a study which concludes that only a STAT1 knockout mouse is susceptible to Reston Ebola virus 6. This strongly points towards the lack of immunosuppressive properties of the Reston Ebola virus in mice.We have also made the suggested minor corrections, and had the manuscript corrected for typographical errors (Mary Mendum has been acknowledged). We hope that we have addressed your concerns by the changes that we have made.Thanking you,Sincerely,Sandeep Chakraborty (Corresponding author) References 1. Chakraborty S, Rao B, Dandekar A: PAGAL - Properties and corresponding graphics of alpha helical structures in proteins [v2; ref status: indexed, http://f1000r.es/4e7]. F1000Research. 2014; 3 (206). PubMed Abstract \| Publisher Full Text \| Reference Source 2. Chakraborty S, Phu M, Rao B, Asgeirsson B, et al.: The PDB database is a rich source of alpha-helical anti-microbial peptides to combat disease causing pathogens [v1; ref status: awaiting peer review, http://f1000r.es/4sa]. F1000Research. 2014; 3 (295). Publisher Full Text \| Reference Source 3. Zhang AP, Abelson DM, Bornholdt ZA, Liu T, et al.: The ebolavirus VP24 interferon antagonist: know your enemy. Virulence. 2012; 3 (5): 440-445 PubMed Abstract \| Free Full Text \| Publisher Full Text \| Reference Source 4. Ebihara H, Takada A, Kobasa D, Jones S, et al.: Molecular determinants of Ebola virus virulence in mice. PLoS Pathog. 2006; 2 (7): e73 PubMed Abstract \| Free Full Text \| Publisher Full Text \| Reference Source 5. Chakraborty S, Rao B, Asgeirsson B, Dandekar A: Characterizing alpha helical properties of Ebola viral proteins as potential targets for inhibition of alpha-helix mediated protein-protein interactions [v2; ref status: approved with reservations 1, http://f1000r.es/4qr]. F1000Research. 2014; 3 (251). Publisher Full Text \| Reference Source 6. de Wit E, Munster VJ, Metwally SA, Feldman H: Assessment of rodents as animal models for Reston ebolavirus. J Infect Dis. 2011; 204 (Suppl 3): S968-S972 PubMed Abstract \| Free Full Text \| Publisher Full Text \| Reference Source" } ] } ]	1	https://f1000research.com/articles/3-265
https://f1000research.com/articles/3-133/v1	24 Jun 14	{ "type": "Research Article", "title": "An evolutionarily significant unicellular strategy in response to starvation stress in Dictyostelium social amoebae", "authors": [ "Darja Dubravcic", "Minus van Baalen", "Clément Nizak", "Darja Dubravcic", "Minus van Baalen" ], "abstract": "The social amoeba Dictyostelium discoideum is widely studied for its multicellular development program as a response to starvation and constitutes a model of choice in microbial cooperation studies. Aggregates of up to 106 cells form fruiting bodies containing two cell types: (i) dormant spores (~80%) that can persist for months in the absence of nutrients, and (ii) dead stalk cells (~20%) that promote the dispersion of the spores towards nutrient-rich areas.It is often overlooked that not all cells aggregate upon starvation. Using a new quantitative approach based on time-lapse fluorescence microscopy and a low ratio of reporting cells, we have quantified this fraction of non-aggregating cells. In realistic starvation conditions, up to 15% of cells do not aggregate, which makes this third cell fate a significant component of the population-level response of social amoebae to starvation. Non-aggregating cells have an advantage over cells in aggregates since they resume growth earlier upon arrival of new nutrients, but have a shorter lifespan under prolonged starvation. We find that phenotypic heterogeneities linked to cell nutritional state bias the representation of cells in the aggregating vs. non-aggregating fractions, and thus regulate population partitioning. Next, we report that the fraction of non-aggregating cells depends on genetic factors that regulate the timing of starvation, signal sensing efficiency and aggregation efficiency. In addition, interactions between clones in mixtures of non-isogenic cells affect the partitioning of each clone into both fractions. We further test the evolutionary significance of the non-aggregating cell fraction. The partitioning of cells into aggregating and non-aggregating fractions is optimal in fluctuating environments with an unpredictable duration of starvation periods. D. discoideum thus constitutes a model system lying at the intersection of microbial cooperation and bet hedging, defining a new frontier in microbiology and evolution studies", "keywords": [ "Every organism has a set of optimal conditions that maximizes its fitness (growth", "reproduction and survival). Yet", "living environments typically deviate from these conditions. In some cases individuals can adapt to changes by sensing the environment and modifying their phenotypes accordingly", "which is known as phenotypic plasticity1. However", "if the sensing mechanism is too costly", "phenotypic plasticity may not be optimal even in the presence of environmental variation. Differentiation on a stochastic basis into different phenotypic states adapted to different environments", "also known as risk spreading or bet hedging", "has also been proposed as an adaptation to environmental variation2–6. Dormant states have often been described as such bet hedging strategies. Examples include plant seed dormancy7", "arthropod diapauses8 and bacterial sporulation9. For entering and exiting the dormant state", "cells or organisms depend on environmental cues. Yet", "these cues are not always reliable indicators of the future environment. Therefore", "in such unpredictable environments it pays off for a plant", "for instance", "to have its seeds germinating stochastically at different time scales to insure that at least some of them will germinate at the time that is beneficial for its growth7." ], "content": "Introduction\n\nEvery organism has a set of optimal conditions that maximizes its fitness (growth, reproduction and survival). Yet, living environments typically deviate from these conditions. In some cases individuals can adapt to changes by sensing the environment and modifying their phenotypes accordingly, which is known as phenotypic plasticity1. However, if the sensing mechanism is too costly, phenotypic plasticity may not be optimal even in the presence of environmental variation. Differentiation on a stochastic basis into different phenotypic states adapted to different environments, also known as risk spreading or bet hedging, has also been proposed as an adaptation to environmental variation2–6. Dormant states have often been described as such bet hedging strategies. Examples include plant seed dormancy7, arthropod diapauses8 and bacterial sporulation9. For entering and exiting the dormant state, cells or organisms depend on environmental cues. Yet, these cues are not always reliable indicators of the future environment. Therefore, in such unpredictable environments it pays off for a plant, for instance, to have its seeds germinating stochastically at different time scales to insure that at least some of them will germinate at the time that is beneficial for its growth7.\n\nHere we focus on the dormancy of the cellular slime mold Dictyostelium discoideum as an adaptation to nutritional stress. D. discoideum amoebae live in soil where they feed on bacteria and divide mitotically. When starved, cells enter into the dormant social phase of the life cycle. Up to 106 cells aggregate to form a multicellular organism that goes through a “slug” stage followed by the formation of a fruiting body. The slug is a motile, chemotactic and phototactic worm-like structure that senses and moves towards environments that are favorable for dispersion, germination and cell proliferation. The fruiting body is a sessile mushroom-like structure with the spore mass sitting on top of a stalk. Dormant spores can survive for months in the absence of food, and germinate into single cells upon dispersion towards nutritive areas. The stalk lifts the spores from the ground, which helps spore dispersion. Cells in the stalk, which represent ~20% of the total cell population, die owing to the metabolic cost of making up the stalk10.\n\nIts social behavior has made D. discoideum a very popular system for studying altruism, cheating and cooperation11,12, but not all aspects of its population-level adaptation to stress have been studied. Our main motivation was to study a previously known but neglected fact that not all cells aggregate upon starvation. We have thus revisited the D. discoideum population-level response to nutritional stress by focusing on the aggregation stage. Incomplete aggregation may have significant evolutionary consequences. Aggregation is costly due to the death of stalk-forming cells and the arrest of cell division during fruiting body formation, which is an irreversible process13. Cells that do not aggregate do not pay these costs and may have the advantage of resuming growth immediately upon arrival of new nutrients. If conditions improve quickly, non-aggregating cells thus may have an important adaptive advantage. While often considered an experimental error or just insignificant, we asked whether the fraction of non-aggregating cells constitutes an important component of the adaptive response to stress.\n\nIn this study we present the first attempt to describe the D. discoideum response to starvation stress as a functional partitioning into two states: aggregating and non-aggregating. We focus on two major points: (i) establishing the phenotypic and genotypic sources of population partitioning and (ii) assessing the evolutionary significance of such partitioning. In microbial systems, cell states such as cell cycle phase, nutritional state or age are sources of phenotypic heterogeneities9,14. Besides, different genetic backgrounds could give rise to different degrees of heterogeneity, giving insights into underlying molecular mechanisms. Here we develop a new technique based on quantitative live cell microscopy to analyze the effects of cell nutritional state, genetic background and environmental organization on population partitioning between aggregating and non-aggregating cells. In addition, we propose a model based on experimentally determined parameters to illustrate the potential evolutionary significance of population partitioning in fluctuating environments.\n\n\nMaterials and methods\n\nD. discoideum axenic strains used in the study were AX3 (Dictybase ID: DBS0235545), DH1 (Dictybase ID: DBS0302388), phg2 (Dictybase ID: DBS0302388), pdsA (Dictybase ID: DBS0237030), and carA (Dictybase ID: DBS0236438). All the strains were cultured in autoclaved HL5 medium (per L, 5 g proteose peptone, 5 g thiotone E peptone, 5 g yeast extract, from USBIO, 10 g glucose, 0.35 g Na2HPO47H2O, 0.35 g KH2PO4 from Sigma-Aldrich, pH=6.7) at 22°C if not mentioned otherwise. In experiments on nutritional effect we used: FM minimal medium (Formedium), NS (per L, 15.2 g peptone, 7.6 g yeast extract, from USBIO, 5mg Na2HPO4, 5mg KH2PO4, from Sigma-Aldrich, pH=6.7) and NS with 85mM glucose (Sigma-Aldrich) added after autoclaving15. The bacterial species used as the nutritional source in our study was Klebsiella aerogenes. Heat killed bacterial cultures were prepared by centrifuging 50mL of overnight LB cultures at 4°C, 5000 g for 10min and diluting the pellet in 1mL KK2 buffer (per L, 22 g KH2PO4, 7.0 g K2HPO4, Sigma-Aldrich). The suspension was incubated for 20min at 80°C and stored at −20°C.\n\nGFP and RFP-expressing cell lines were obtained by transforming cells with pTX-GFP (Dictybase ID: 11)16 or pTX-RFP (Dictybase ID: 112) plasmids using a standard electroporation procedure. Cells were grown in 75cm2 flasks until dense but not confluent (usually 1 day before confluency). The medium was changed 4–6h before transformation. For transformation cells were re-suspended in 10mL of ice-cold HL5 and kept on ice for 30min. Cells were centrifuged for 5min, 500 g at 4°C. Supernatant was re-suspended in 800μl of electroporation buffer and transferred into ice cold 4mm electroporation cuvettes containing 30μg of plasmid DNA. Cells were electroporated at 0.85 kV and 25 mF twice, waiting for 5 s between pulses. Cells were transferred from the cuvette to 75cm2 flask with HL5. The next day, transformants were selected with 5μg/ml G418 (Sigma-Aldrich). The concentration of G418 was gradually increased to 20μg/ml G418 over 1–2 weeks. Transformed strains were maintained at this concentration of G418, yielding GFP and RFP-expressing cell lines that were analyzed by flow cytometry on a Becton-Dickinson LSRII analyzer to confirm their unimodal cellular fluorescence distribution (>99% of fluorescent cells upon analysis of 106 cells, see Supplementary Figure 6).\n\nCells were subjected to two different starvation conditions: sudden and gradual starvation. For each condition measurement was repeated 4–11 times (see Raw data for further details, each measurement is an independent experiment).\n\nSudden starvation: If not mentioned otherwise, sudden starvation was used as a standard plating protocol: When confluent the cell medium with antibiotics was replaced with an antibiotic free medium. After 4–6h cells were washed out of the nutrient medium and centrifuged in KK2 buffer at 500g for 5 min. The pellet was re-suspended in KK2 buffer to the concentration of 1×105 cells/μL. For the density dependent aggregation experiment cells were re-suspended to the concentration of 1×103, 1×104, 5×104, 1×105 or 5–7.5×105 cells/μL. Green and red fluorescent cells were mixed in ratios indicated in Image analysis section. 30μl of suspension was plated on 6cm plates filled with 2mL of 2% Phytagel (Sigma-Aldrich) as previously described17. In the case of pairwise mixtures, strains grown in different media or genetically different strains, the ratio of two strains was 1:1.\n\nGradual starvation was induced in liquid cultures and on bacterial plates.\n\nGradual starvation in liquid: the cells were collected 1–2 days after reaching confluency in HL5. Cell washing and plating was done as in sudden starvation experiment described above.\n\nGradual starvation on bacterial plates: another way of slowly starving the cells is to plate them with bacteria and to let them deplete the food source as in natural conditions. Two types of plating were done: homogenous and heterogeneous plating. In both cases RFP-expressing AX3 and GFP-expressing AX3 cells were grown in HL5 medium with 20μg/mL G418. When confluent, cells were re-suspended in KK2 buffer and centrifuged at 500 g for 5min. The cell pellet was re-suspended in KK2 to the concentration of 1×105 cells/μL. Green and red fluorescent cells were mixed in ratios indicated in Image analysis section. For heterogeneous plating 200μL of heat-killed bacteria was mixed with 100μl of cell suspension. The mixture was spread on a 6cm plate with 2mL of 2% Phytagel (Sigma-Aldrich). This gave rise to heterogeneous distribution of cells and bacteria (Supplementary Figure S2). For homogenous plating 200μl of heat-killed bacteria were mixed with 100μl of cell suspension. A 100μl drop was plated on a 6cm plate with 2ml of 2% Phytagel and let to dry under the sterile hood. This gave a very homogeneous cell distribution (Supplementary Figure S2). In both cases, cells fed for ~8h on heat-killed bacteria before the beginning of starvation, and thus divided at most twice after plating. The density of cells at the onset of starvation (measured via a similar method as the one for measuring the non-aggregating cell fraction, see below) was comparable to that of cells processed according to the sudden starvation protocol.\n\nThe 6cm diameter Petri dish was imaged on an automated inverted microscope setup duplicated from a previous study18. The setup was made of: OlympusIX70 inverted microscope, Photometrics CoolSNAP HQ2 CCD camera, Zeiss HBO 100 microscope illuminating system, Thorlabs SH05 shutter, Thorlabs TSC001 shutter controller, and 2.5×-5×-10×-20× objectives (5× was used for all experiments shown here). Images were acquired in WinView/32 and the whole setup was controlled by custom-made Visual Basic software. The setup allows Petri dish scanning at regular time intervals (typically 1h), with phase contrast and fluorescence image acquisition at all time points (at 100ms and 1s exposure times respectively). A mosaic image is reconstructed by combining all the images of contiguous areas of the Petri dish at a given time point by a custom-made macro using ImageJ software (http://rsbweb.nih.gov/ij/).\n\nMixing a small percentage of red fluorescent cells in a population of green fluorescent cells allowed us to get the image of single cells as single red fluorescent dots (Figure 1). We also confirmed that the reciprocal mixing of a minority of GFP-expressing cells with a majority of RFP-expressing cells yields the same results. We optimized the red to green cell ratios depending on plated cell density. For experiments with 1×105 and 5–7.5×105 cells/μL, 0.25–0.5% of RFP cells were mixed with 99.5–99.75% GFP cells. For 1×104 cells/μL 1% of RFP cells were used and for 1×103 cells/μL 2% RFP cells were used. For pairwise mixtures the ratio was made as following: 50% of strain A in GFP was mixed with 49.75% of strain B in GFP and 0.25% of strain B in RFP in order to monitor the behavior of strain B in a A:B mixture. Images were acquired by time-lapse fluorescence microscopy. All the images were analyzed using ImageJ software (http://rsbweb.nih.gov/ij/) using custom-made macros (see Data Set). The analysis consisted in counting fluorescent dots before and after aggregation. For each experiment 1000–10 000 dots/cells were monitored. Dead cells were excluded from counting by looking at cell displacement as an indicator of cell viability. Two fluorescent images taken 1–2h apart were overlapped and cells that showed no displacement were counted and subtracted from the overall non-aggregating population.\n\nAX3 cells were plated on nutrient free-agar and imaged before (A, B) and after (C, D) aggregation. A and C are phase contrast images, B and D are red fluorescence images. In B and D, 0.25% of AX3 RFP cells appear as single dots within a population of AX3 GFP cells. The percentage of non-aggregating cells was estimated as the ratio of dots counted outside aggregates after aggregation and dots counted before aggregation.\n\nThe density of red dots (RFP-expressing cells) was used to estimate cell density at the onset of starvation in all experiments. Cell density was comparable at the onset of starvation for all starvation protocols used.\n\nSpore formation was induced by separately plating AX3 RFP and AX3 GFP cells on nutrient-free Phytagel plates. Once fruiting bodies had formed spores were picked using 1ml pipette tips and re-suspended at high density in 5m liquid HL5 with 70μL of dead bacteria culture. Presence of bacteria helped to induce spore germination. When the culture of germinated spores reached confluency (15–20h after plating) cells were washed of bacteria in ice cold KK2 and plated according to the Sudden starvation protocol.\n\nThe model represents the D. discoideum life cycle with alternating growth and starvation periods of variable duration. During the growth phase the population grows according to a logistic equation (1) with growth rate λ and carrying capacity K = Nmax,\n\n\n\nWe assume that the growth phase lasts sufficiently long for the population to have reached maximum density K when the food eventually runs out and a starvation period T sets in. The population then splits into an aggregating (Nagg = αN) and a non-aggregating (Nnon -agg = (1–α)N) fraction according to the aggregation factor α. Aggregating cells subsequently differentiate into spore and stalk cells with the proportion of spore cells given by sporulation efficiency s, so Nspores = sNagg. We assume the process of aggregation is very quick relative to the duration of the growth and starvation periods. During the starvation period spores are dormant; their growth and mortality rate are assumed to be zero. When conditions become favorable again, spores germinate with germination efficiency g and start dividing, but only after a fixed and non-negligible development time D. During the starvation period the non-aggregating cells do not divide and are subjected to mortality with instantaneous mortality rate μ, so that their dynamics are governed by\n\n\n\nThe advantage that non-aggregating cells have is a head start when conditions improve, as spores produced by aggregating cells need time to develop. By the time the latter start growing, the descendants of the non-aggregating cells may have the opportunity to use up a sizable portion of the resources that have become available. Here, we assume that spore germination is limited by the remaining carrying capacity.\n\nAs a first step in understanding the relative benefits of aggregation and non-aggregation consider the fates of cells of either type at the moment starvation sets in. A non-aggregating cell stops reproducing but is subject to mortality so when conditions become favourable again, T time units later, it has a probability e-μT of surviving the starvation period. Working out the fate of aggregating cells is simple: it has a probability sg of becoming a germinating spore when conditions improve. An aggregating cell thus has a fitness equivalent of\n\n\n\nAs discussed, germination involves a time cost: during a time D its surviving non-aggregating competitors can start reproducing, giving the latter an extra reproduction bonus (a period of logistic growth), giving a fitness equivalent of\n\n\n\nwhere n0 is the number of surviving non-aggregating cells.\n\nThe expected fitness (descendants by the time conditions improve) of a cell that has a propensity α to aggregate can thus be expressed as\n\n\n\nThis result suggests that (if the duration of the starvation period is fixed) it is either profitable to join an aggregation (if Wagg > Wnon–agg) or to stay solitary (if Wagg < Wnon–agg): a bet-hedging strategy is not favored. However, this result does not take into account the frequency dependence that acts on the fitness of non-aggregating cells. That is, if many cells aggregate the number of surviving non-aggregating cells (n0) will be low, boosting the profitability of remaining solitary. If many cells remain solitary, on the other hand, n0 will be high, reducing the profitability of remaining solitary. Whether this frequency dependence results in population heterogeneity cannot be stated right away and other methods are necessary. The same is true when the environment, and in particular the starvation period T, is variable and unpredictable.\n\nIn order to study potential benefits of producing both aggregating and non-aggregating cells, strains with different aggregation factors α were put in competition using a multistrain variant of the above-described model. The population is made of i strains each with α=0 (all cells aggregate), 0.1, 0.2, … 1 (none of the cells aggregate). All strains had the same growth rate λ = 0.38, mortality rate μ = 0.002 for t ≤ 168h (7 days), after 7 days all cells die, μ = 0, sporulation efficiency s = 0.8 and germination efficiency g = 0.63. All values are based on experimental measurements (Materials and Methods in Supplementary materials). Two-step mortality function is an approximation based on our unpublished results and previous studies19,20. The precise shape of this function had no significant effect on our main observations and conclusions. Competition was carried out in two types of conditions, either constant or varying starvation periods T. In the case of varying starvation periods, the duration of starvation was randomly chosen from a uniform distribution U(x,y) at the end of every growth period. Population size was taken as an estimate of strain fitness. At the end of every growth cycle, the number of alive and growing individuals N(t) is plotted. In the case of varying starvation periods, the geometric mean over 100 simulations is plotted.\n\n\n\nStatistical analysis was performed in R. Significant difference between the samples was calculated using Welch two sample t test function in R (t.test(x,y)). To test among groups differences we used one-way ANOVA test in R, using oneway.test() function. When only p value is indicated it means that a t-test was performed, when p and F values are indicated ANOVA was performed. P<0.05 was considered significant.\n\n\nResults\n\nWhen we plated a population of genetically identical axenic wild-type AX3 cells of D. discoideum on nutrient-free substrates at a 104–107 cells/cm2 density range21, we observed that some cells aggregate while others remain outside of aggregates (Figure 1, Supplementary Figure S1). A possible explanation is that the cells that did not aggregate are simply dead cells. However, the observation that non-aggregating cells are actively moving, live cells that are intermixed with aggregating cells at the onset of starvation (Movie S1 in the Data Set below) rules out this possibility. It could also be that these non-aggregating cells have acquired a mutation that prevents aggregation. As we will detail further, this possibility can be ruled out by showing that the progeny of spores are partially non-aggregating and reciprocally that the progeny of non-aggregating cells aggregate upon starvation. Another explanation may be that partial aggregation is an artifact of a laboratory-adapted axenic strain that is not found in natural isolates, but in Supplementary Figure S2 we show that similar partitioning is found in natural isolates. Partitioning into aggregating and non-aggregating cells is therefore a process that occurs in both axenic strains and isolates of social amoebae from the wild. The non-aggregating cells we report here are clearly distinct from cells left in slug traces22 since the former never aggregate as we have shown in Movie S1. For the same reason, non-aggregating cells are also clearly distinct from the immune-like cells identified in a previous study23. The motility of the non-aggregating single cells we observe also rules out the possibility that these cells are sporulating without aggregating, as in single cell encystation that has been reported for other Dictyostelium species but not so far in D. discoideum10.\n\nTo quantitatively analyze this process, we have developed a technique to track single cell behavior at each time point of the life cycle. Inspired by studies of cell motion within aggregates24, a small proportion (0.25%–2%) of RFP-expressing reporting cells was mixed with GFP-expressing cells, and RFP cells were tracked (see Materials and methods). In the red fluorescence image single RFP cells appear as single red dots surrounded by undistinguishable GFP cells (Figure 1B). Since cell division ceases during starvation, tracking RFP-expressing single cells allowed us to determine the relative numbers of aggregating vs. non-aggregating cells, and thus to quantitatively describe the population partitioning into aggregating and non-aggregating cells. Previous techniques based on counting cells at the onset of starvation with a hemocytometer and germinating/colony-forming spores provide only indirect estimation of the numbers of stalk cells, non-aggregating cells, or non-germinating spores. In contrast, our strategy provides a direct estimation of the numbers of cells at the onset of starvation and aggregating vs. non-aggregating cells. Our automated microscopy setup is similar to the one used in a previous study of large scale population spatial structure at the single cell resolution18. We scan and image by phase contrast and fluorescence microscopy an area of 5cm2 every 10min for 24h, allowing us to record the dynamics of the response of large populations (millions of cells) at the single cell resolution.\n\nUsing our set-up, we found that when cells of the AX3 wild-type axenic strain are grown in liquid rich medium (HL5) and subsequently plated on nutrient-free substrate, 2.51±0.6% of the population does not aggregate. This standard starvation protocol involves the sudden transition from exponential growth in rich medium to starvation on nutrient-free agar. However, in natural conditions starvation is probably much more gradual. We analyzed how different starvation processes can affect population partitioning (Figure 2A) at the same cell density range at the onset of starvation. We compared (i) suddenly starved exponentially growing cells, (ii) starved stationary phase cells (1–2 days after confluency), and (iii) cells grown on bacterial plates that slowly deplete the food source, the latter being the most realistic starvation process with respect to natural conditions. While stationary phase cells show no significant difference compared to exponentially growing cells, cells feeding on a homogenous bacterial lawn and thus gradually starving showed a 3-fold increase in the proportion of non-aggregating cells, 6.3±3.17% (p=0.027).\n\nThe percentage of non-aggregated cells (at initial density 3×106 cells/cm2) was measured for different initial cell states. A) Effect of starvation conditions. AX3 RFP and GFP cells were starved suddenly at exponential phase or at stationary phase, or gradually on homogenous bacterial lawns or on heterogeneous bacterial lawns. Gradually starved cells aggregate less than cells submitted to standard but less realistic sudden starvation protocols. B) Effect of nutritional state. AX3 cells were grown on HL5 rich medium, FM minimal medium, NS with 85mM Glucose (NS Glu) or NS medium, and subsequently plated on nutrient-free agar. Cells in the lowest nutritional state (FM) aggregate significantly less than cells fed with rich medium. C) Interactions between cells in different nutritional states. AX3 cells grown on HL5 or FM were plated either on their own or in 1:1 mixtures on nutrient-free agar (HL5inFM = HL5 cells monitored in 1:1 mixtures, and FMinHL5 = FM cells monitored in 1:1 mixtures). In mixtures with HL5-grown cells, FM-grown cells aggregate even less than on their own, while HL5-grown cells aggregate equally well in the presence of FM-grown cells as on their own. Note that the non-aggregating cell fraction of the global mixed population is higher than that of either pure population. Error bars represent +/- standard deviation. represents p<0.05. ** represents p<0.01.\n\nGradual starvation on bacterial plates most likely increases heterogeneities in comparison with standard starvation protocols. We supposed that this was due to cell-to-cell differences in the timing of starvation. Some cells would start aggregating while others were not yet fully starved and therefore less sensitive (or not at all) to the aggregation signal. Increasing further heterogeneities during cell plating should thus increase further the non-aggregating cell fraction. This is indeed the case when a heterogeneous bacterial lawn is used as a food source, where the fraction of non-aggregating cells increases to 13%±1.79% (p=0.004). A possible explanation is that highly heterogeneous cell plating creates areas with different cell densities within a lawn of bacteria (Supplementary Figure S3C, D). Areas with high cell densities deplete bacteria faster and start starving and aggregating quicker, while cells in low cell density areas still have nutrients surrounding them and are not sensitive to the aggregation signal when the former sense starvation. In homogenous bacterial lawns, cells and bacteria are evenly distributed favoring more homogenous and synchronous onset of starvation across the population (Supplementary Figure S3). We hypothesized that differences at the onset of starvation result in a cell fate bias towards one phenotype or the other (as previously proposed in the case of stalk vs. spore differentiation in aggregates25). To analyze these effects in the most reproducible and controllable manner, all following experiments were performed following the standard sudden starvation protocol (plating on nutrient-free agar) applied to cells grown in various well-defined conditions, with known genetic backgrounds, mixed at precise ratios and plated at controlled cell densities.\n\nNutritional state is known to affect whether a cell will becomes a spore or a stalk26. Cells grown on rich medium (NS medium with 85mM glucose) are enriched in spores while cells grown in poorer medium (NS medium lacking glucose) are enriched in the stalk (which we have also observed, see Supplementary Figure S5). We thus asked whether nutritional state is a main determinant of the aggregating and non-aggregating dichotomy. We grew AX3 cells in media differing in nutrient content and analyzed whether they are differentially enriched in the non-aggregating state (Figure 2B). Four different media were tested: HL5 rich medium, FM minimal medium, NS with 85mM glucose (NS Glu) and NS medium. AX3 cells grown on FM minimal medium showed a significant two-fold increase in the fraction of non-aggregating cells, 5.85±1.9% (p<0.01), with respect to HL5-grown cells (2.51±0.6%). In addition, cells grown on NS Glu medium showed a small but significant decrease in non-aggregating cells (1.47±0.31%, p<0.01) compared to HL5 grown cells (2.51±0.6%). However, we observed that cells grown in NS medium did not differ from cells grown in NS with glucose in terms of non-aggregating cell fraction, making the role of glucose difficult to interpret.\n\nCells in different nutritional states have different aggregation rates on their own. We next examined how cells in different nutritional states interact in mixtures in order to analyze how introducing population nutritional state heterogeneity affects population partitioning. Pairwise mixtures of FM-grown cells with HL5-grown cells and NS-grown cells with NS Glu-grown cells were tested. Cells grown in NS or NS Glu that did not differ when alone showed no difference in behavior when in mixtures (Supplementary Figure S4) (F=1.54, p=0.27). On the other hand cells grown in FM were enriched 3 times more in the non-aggregating cell fraction when in mixture with HL5-grown cells, 15.4±7.12%, than on their own, 5.85% (Figure 2C). HL5-grown cells aggregated equally well when in mixture with FM-grown cells or not. As a control we monitored contribution to spores for both mixtures. As previously shown, cells grown in rich medium were enriched in spores in both NS Glu:NS and HL5:FM mixtures (Supplementary Figure S5).\n\nWe conclude that nutritional state distinguishes non-aggregating cells from aggregating cells, and that interactions between cells according to their nutritional state biases further partitioning between aggregating and non-aggregation cell fates. Moreover, the 1:1 mixed population of cells having different nutritional status showed a higher fraction of non-aggregating cells than the average of both populations. This is consistent with our data obtained with populations grown on heterogeneous food source showing a higher proportion of non-aggregating cells. Cells grown on low nutrient medium have higher chances of becoming non-aggregated cells than cells grown on rich medium. The fact that NS-grown cells displayed the same behavior as NS Glu-grown and HL5-grown cells is probably because cells were relatively well fed in all three cases and not much affected by the absence of glucose15. On the other hand FM-grown cells showed smaller cell size, slower growth and lower inner cell density indicating that they were affected by growth in poor medium (our unpublished observation). We can speculate that poorly fed FM-grown cells have low energy reserves, and that they consequently invest less into energetically costly multicellular development and thus aggregate less. The fact that, in mixtures with HL5-grown cells, FM-grown cells showed an even lower rate of aggregation indicates the effect of cell-cell interactions during aggregation. No difference in the timing of aggregation was seen between FM- and HL5-grown cells. Therefore, cell nutritional state rather than aggregation timing was the cause of the differences in the fraction of non-aggregating cells.\n\nAfter exploring nutritional state effects, we tested whether different genetic backgrounds can lead to different population partitioning. In Figure 3A we show that two axenic strains, DH1 and AX3, significantly differ in the fraction of non-aggregating cells (p=0.0008). The DH1 strain showed 13.4%±2.8% of non-aggregating cells, which is five times higher than for the AX3 strain (2.5%±0.6%). This shows that the non-aggregating cell fraction depends on the genetic background and varies significantly between axenic wild-type strains.\n\nThe percentage of non-aggregated cells (at initial density 3×106 cells/cm2) was measured for genetically different wild-type strains (AX3 and DH1) and single-gene mutants (phg2, pdsA, carA) alone (A), and in mixtures between wild-type and single-gene mutant strains: mixtures of pdsA with AX3 (B) or DH1 (C), and mixtures of phg2 with AX3 (E) or DH1 (F)., varying the percentage of mutant cells in mixtures. Wild-type DH1 cells aggregate less than wild-type AX3 cells (A). phg2 mutant cells aggregate as well as their parent DH1 strain cells, while pdsA and carA cells do not aggregate on their own (A). The presence of AX3 or DH1 cells rescues pdsA cell aggregation (B–D). In turn, DH1 cells aggregate less than on their own when increasing the percentage of pdsA cells in DH1:pdsA mixtures, while AX3 cells aggregate as well as on their own in the presence of pdsA cells (B–D). phg2 cells aggregate less than on their own in the presence of AX3 or DH1 cells. DH1 cells aggregate less than on their own in DH1:phg2 mixtures, while AX3 cells aggregate as well as on their own in the presence of phg2 cells (E–G). The non-aggregating cell fraction of the global mixed DH1:phg2 or AX3:phg2 population is higher than that of either pure populations, respectively DH1 and phg2, or AX3 and phg2. Overall, cell genotype determines the fraction of aggregating cells, and cells of different genotypes affect each other’s non-aggregating cell fraction in mixtures. Error bars represent +/- standard deviation. * represents p<0.05. *** represents p<0.001.\n\nFollowing these results, we explored which genetic mechanisms may affect the cell propensity for aggregating or non-aggregating fates. For this, we first tested strains with single gene mutations in aggregation pathways. We used two mutants defective in signal sensing: 1) carA, a mutant in cAMP receptor protein cAR1, which is essential for binding the chemo-attractant cAMP and 2) pdsA, a mutant in cAMP-phosphodiesterase (PDE), which removes cAMP from its cAR1 receptor making it sensitive again to the aggregation signal10. Our results confirmed the previously reported result that when plated on nutrient-free agar, both strains showed no aggregation at all (Figure 3A)27,28. This shows how single gene mutations may have a drastic effect on population partitioning. It is known that the presence of wild-type cells can rescue the non-aggregating pdsA phenotype (non-cell autonomous)29. Our technique allows the quantification of aggregation efficiency of mutant and wild-type cells in mixtures. We thus varied the ratio of wild-type cells (AX3 or DH1) in mixtures with mutant pdsA cells from 10% to 90% and quantified how it affects aggregation of pdsA mutant and wild type strains. For both DH1:pdsA and AX3:pdsA mixtures, increasing the ratio of wild type cells decreased the proportion of pdsA non-aggregating cells (Figure 3B–D). Aggregation rescue of mutant cells came at a cost for the DH1 strain; the fraction of non-aggregating cells for DH1 increased in mixtures with pdsA (Figure 3C, D). In AX3:pdsA mixtures, AX3 cells aggregated as much as and pdsA cells aggregated more than when on their own (Figure 3B, D), suggesting that AX3 produces more PDE protein than DH1. More generally, we propose that expression levels of cAMP-phosphodiesterase may tune the non-aggregated cell fraction. Low concentration of cAMP-phosphodiesterase would increase the fraction of non-aggregating cells.\n\nWe found that differences in starvation sensing affect the partitioning between aggregating and non-aggregating fractions (Figure 2A). The phg2 mutant strain has been shown to have early onset of starvation compared to its parental strain DH1 due to a higher nutrient starvation sensing threshold30. We used this single gene mutant to test the effect of the nutrition starvation sensing threshold on partitioning. In addition, the phg2 gene codes for a serine/threonine kinase regulating cell substrate adhesion, actin cytoskeleton organization and motility31. When tested alone, phg2 produced a similar fraction of non-aggregated cells when compared to its parental strain DH1, 12.6%±4.3% (p=0.7). We further tested the behavior of phg2 in 1:1 mixtures with wild-type strains DH1 and AX3. Mixing at 1:1 led to an increase of the non-aggregating cell fraction for phg2 and its DH1 parent (Figure 3F, G), while AX3 aggregated equally well as when on its own (Figure 3E, G). This once more demonstrates that in mixtures, strains mutually affect each other’s non-aggregating cell fractions. Indeed, the phg2 mutant aggregates less in 1:1 mixtures with wild-type cells than on its own, even in mixtures with its parent DH1 wild-type strain that has a similar aggregation fraction on its own. Moreover, in 1:1 mixtures of phg2 with DH1 or AX3, the global mixed population shows a significant increase in the fraction of non-aggregating cells with respect to both pure populations. This is again reminiscent of our previous results that population heterogeneities in nutritional state (cells grown on heterogeneous bacterial lawns, or on HL5 vs. FM) increase the non-aggregation fraction of the global population. In addition to starvation sensing, the dysfunctional cytoskeleton organization and motility of the phg2 strain could explain the lower propensity of phg2 cells for aggregation.\n\nWe further tested whether non-aggregation is due to a mutation or a bet hedging-like strategy between aggregation and non-aggregation. Can the same population partitioning be reproduced by starting from only aggregating or only non-aggregating cells? Answering this question allows us to: i) rule out any genetic differences between aggregating and non-aggregating cells and ii) examine the effect of epigenetic inheritance of cell fate. When non-aggregating cells are de novo fed with bacteria, they resume growth on new nutrients (see below) until they are exhausted and finally aggregate upon starvation (Figure 4A–C and Movie S2). This shows that non-aggregating cells are not mutant cells that cannot aggregate, but rather cells that are not responding to the aggregation signal at a given time point. Further on in Figure 4D we show that a population of germinated spore cells dividing 3 to 5 times upon germination partitions into aggregating and non-aggregating cells with the same fractions as a population of exponentially growing cells. This demonstrates the strong persistence of population partitioning and the fast loss of cell epigenetic memory.\n\nA–C Non-aggregating cells have not lost the genetic ability to aggregate. A) After the completion of aggregation and formation of fruiting bodies (white arrows), bacteria were added to areas with non-aggregating cells (black arrows). Non-aggregating cells grow and divide on fresh nutrients (see Figure 5). Once bacteria are consumed, the descendants of non-aggregating cells aggregate (B) and develop into a fruiting body (C). D) A population of germinated spores re-partitions into aggregating and non-aggregating cells upon starvation. A population of spores was germinated and grown on bacteria for 3–5 cell divisions. When this population is plated on a nutrient-free substrate it partitions into aggregating and non-aggregating cells with the same proportions as populations of exponentially growing cells submitted to starvation.\n\n18h after plating cells on nutrient-free agar, aggregating cells have formed slugs while non-aggregating cells are starving. Fresh nutrients (dead bacteria) were added at this point. A) Red fluorescence image of slugs and non-aggregating cells at the time of new nutrient supply. B) Inset from A showing a non-aggregating cell that resumes dividing over time upon addition of new nutrients (the number of red dots increases over time as non-aggregating cells divide). Non-aggregating cells are capable of resuming growth immediately upon food arrival while aggregating cells are embedded in development.\n\nWe have shown that upon starvation D. discoideum cell populations partition into cells that aggregate and cells that do not aggregate, and that non-genetic and genetic cell characteristics affect cell fates. We next analyze evolutionary consequences of this population partitioning. To do this we analyzed fitness costs and benefits of both phenotypes on individual and population levels.\n\nOnce in an aggregate a cell is irreversibly committed to the multicellular development program13. During the 24h duration of development, cells cannot divide even if nutrients become available. Therefore, if food becomes available during the developmental period, non-aggregating cells may have an advantage over aggregating cells by immediately resuming growth. We tested this by adding a bacterial suspension to a starving D. discoideum population during the course of development. At this point aggregates were at the slug stage and non-aggregating cells in their vicinity had direct access to food (Figure 5A). In Figure 5B and Movie S3 we show that non-aggregating cells are capable of resuming cell division directly after arrival of nutrients, while slugs (formed of non-dividing aggregated cells) continue moving through the bacterial lawn and form fruiting bodies. Our observation is clearly distinct from previous reports describing the dedifferentiation and re-growth of cells from artificially disaggregated slugs put in contact with fresh nutrients32 since non-aggregating cells do not originate from slugs and are therefore not differentiated into prespore or prestalk. We also observed that by the time fruiting bodies are formed, non-aggregating cells have already consumed a high amount of nutrients, which will probably affect spore fitness by limiting the resources available for spore germination and proliferation (Movie S3).\n\nNon-aggregating cells are motile and do not seem to enter a dormant state like spores do, making them likely to be much less fit than spores during prolonged starvation. Previous studies have reported starvation-induced mortality curves showing that most cells survive for 4 to 7 days19 (corroborated by our unpublished results). These studies demonstrated that, in the absence of food, cells survive through autophagy, degrading their own cytoplasmic components and organelles. Once cells have degraded most of the inner cell components and autophagy can no longer serve as a mode of survival, mortality rate increases and cells die within a day. Non-aggregating cells are expected to pay the same survival costs during long starvation periods.\n\nTo test how phenotypic partitioning affects population fitness, we developed a mathematical model that mimics the D. discoideum life cycle. We asked whether particular non-aggregation rates are selected in fluctuating environments having different, constant or variable, starvation duration and frequency. The model was defined as follows. Not all cells aggregate (Figure 1), cells that do not aggregate die at a defined mortality rate19 (and our unpublished results), non-aggregating cells are capable of resuming growth upon arrival of bacteria (Figure 5A and B, Movie S3); once in an aggregate cells do not divide and are committed to multicellular development until the end13. All the parameters used in the model, such as growth rate, sporulation efficiency and germination efficiency were measured experimentally (see Supplementary materials). Since aggregation is an adaptation to starvation and since the duration of starvation affects costs and benefits of each phenotype (mortality, growth), we tested how the duration of starvation determines the optimal non-aggregating rate.\n\nWe defined 11 strains differing in their non-aggregating cell fractions and calculated their geometric growth rate as a fitness measure. Investment into non-aggregating cells ranged from all cells aggregate (value 1) to none of the cells aggregate (value 0) and was fixed for each strain during the whole competition. For the sake of simplicity, we did not take into account interactions between strains that may increase or decrease aggregation rates, even though our experimental results demonstrated that such interactions do occur and that heterogeneities play a role. In Figure 6A and B we show that under constant starvation periods there are two stable strategies: no aggregation for starvation periods under seven days (168h), and complete aggregation for longer starvation periods. The switch point at 168h is due to the 100% mortality rate after this period. Use of different mortality rates and functions did not significantly change the results (the time period for each optimal strategy just shifted). Since natural environments are rarely constant, with only long or only short starvation periods, we tested competition in environments with fluctuating, long (>168h) and short (<168h) starvation periods. We find that population partitioning into both aggregating and non-aggregating cells gives the highest (geometric) fitness benefits in these fluctuating conditions (Figure 6C and 6D). The results also show that different fluctuations in starvation duration select for different non-aggregating rates. This is in agreement with other models and experiments that showed that optimal population response depends on the rate of environmental fluctuations2,3,33.\n\nEleven strains with different fixed investments into non-aggregating cells were competed under different starvation conditions. Strain investment into non-aggregating cells varies from 0 to 1, with 1 corresponding to complete aggregation and 0 to no aggregation. The duration of the starvation period was varied from <168h (A), >168h (B), randomly taken between 10h and 200h (C), randomly taken between 10h and 300h (D). For systematically long (>168h, B) and short (<168h, A) durations of starvation, strains with 100% aggregation and 0% aggregation take over respectively. For random starvation duration, a particular aggregation rate is selected, for instance 0.4 for 10h<T<200h (C) and 0.9 for 10h<T<300h (D), and thus the superimposition of both strategies is the optimal response.\n\n\nDiscussion\n\nWe report that upon starvation stress a population of D. discoideum amoebae partitions into the widely studied multicellular structures (consisting of live but dormant spores and dead stalk cells) and a fraction that remains unicellular (non-aggregating cells). We have measured the fraction of non-aggregating cells and found that it can amount to up to 15% of the total population in realistic starvation conditions. This is much higher than the 2–3% of non-aggregating cells that result in the standard sudden starvation protocols, and shows that it is important to mimic natural conditions. Non-aggregating cells are live (Supplementary Movie S1), non-mutated cells (Figure 4) that occur in both axenic strain and natural isolates (Supplementary Figure S2). We have thus demonstrated that the non-aggregating cell fraction in natural starvation conditions constitutes a significant component of the population-level starvation response, at least of the order of the stalk cell subpopulation. For our detailed analysis of genetic and non-genetic contributions, we have nevertheless employed the standard sudden starvation protocol to ensure full control over cell population composition and nutritional state, even though this protocol tends to minimize the non-aggregating cell fraction.\n\nIn isogenic populations, we show that partitioning depends on phenotypic heterogeneities linked to cell nutritional state. This is a previously reported determinant of the differentiation between spore and stalk cell fate in aggregates26, together with intracellular Ca2+ levels34 and cell cycle phase35. Decreased aggregation in cells with low nutritional status correlates with lower investment into energetically costly aggregation. The nutritional state-dependent partitioning of the social amoebae population is reminiscent of previous studies reporting non-genetic population heterogeneities in Escherichia coli persistor strains36, Pseudomonas fluorescens colony morphology37, Bacillus subtilis sporulation9 and many others.\n\nDifferent genetic backgrounds can give rise to different levels of heterogeneity9,14,25,38, giving insights into the underlying molecular mechanisms. We demonstrate that genetically different wild-type strains show different non-aggregating cell fractions. This has important implications when drawing a parallel with natural conditions. Distinct genetic strains in nature may show different aggregation fractions leading to competition between different aggregation strategies, as we explore in our model in Figure 6. Further, our results on single-gene mutants underlie possible mechanistic differences between aggregated and non-aggregated cells. We propose that genetic factors that regulate the timing of starvation, signal sensing efficiency and aggregation efficiency largely determine whether a cell adopts the aggregating or non-aggregating phenotype. We confirm that cAR1 and pdsA mutants (Figure 3A and 3B), which are deficient in signal sensing, clearly display non-aggregating cell fractions that differ from their parent strain. Differences in gene expression levels are a known source of phenotypic heterogeneities; comK in B. subtilis cell competence39, spoA in B. subtilis sporulation9, Saccharomyces cerevisiae FLO-dependent phenotype40. It would be very interesting to monitor the same for early developmental genes, expressed at the beginning of aggregation, to see if distinct expression levels correlate with aggregating and non-aggregating cell fates. Genes that control the efficiency of aggregation such as cAR1 and pdsA are potential candidates.\n\nOur results on interactions between mutant and wild type cells in mixtures show that partitioning of social amoebae populations is a complex process, and that competition between genotypes with different aggregation rates is non-linear. In other words, the behavior of strains in mixtures is not the mere linear superposition of their behaviors when on their own, which is reminiscent of the well-documented behavior of strains in mixtures during sporulation experiments41–43. Importantly, even if certain mutants such as phg2 (starvation sensing and motility mutant) do not display a fraction of non-aggregating cells that differs from their parent strain, the non-aggregating cell fraction of the global population may increase (Figure 3) as a result of heterogeneities as is the case of cells grown in heterogeneous conditions (Figure 2, Supplementary Figure 3). We propose that population heterogeneities, due to both genetic and phenotypic causes, play a key quantitative role in population partitioning between unicellular and multicellular cell fates. The effects of nutrition status heterogeneities we report are reminiscent of the previously reported link between nutrition status, cell cycle or Ca2+ content heterogeneities and prespore vs. prestalk differentiation. In nature, social amoebae gradually deplete their food source and spatial distributions of genetic clones largely overlap, thus making both phenotypic and genetic heterogeneities realistic causes of the unicellular vs. multicellular starvation response we describe, and hence reinforcing the ecological significance of our findings.\n\nDifferent phenotypes are often associated with different fitness costs and benefits. In our case, dormant spores survive for months without nutrients but take advantage of incoming food with a delay in comparison to non-aggregating cells. This lag corresponds to the duration of multicellular development and germination, up to 30h or 8 times the single cell division time. Therefore, non-aggregating cells may divide up to 8 times when nutrients are present soon after the beginning of multicellular development, while aggregating sporulating cells do not divide until the end of germination (Figure 5). This confers a considerable evolutionary advantage to non-aggregating cells in such situations (28=256-fold). Our model explores the long term, evolutionary consequences of these effects on the competition between clones with different aggregation rates in fluctuating environments. We find that the aggregation rate is under selection in fluctuating environments and that the optimal rate depends on fluctuations in starvation duration and frequency.\n\nStrategies in which different phenotypes may show differential fitness advantages in different environments are often called bet hedging, and have been shown to be adaptive in fluctuating environments2–6,37. In plants, the success of germination often depends on precipitation. Since rainfall is unpredictable and variable, the diversification of germination timings within season was predicted and demonstrated7. Similar examples include mosquito egg hatching44, copepod egg diapause45, phenotypic switching in S. cerevisiae3, persistor phenotype in E. coli33 and many others7. B. subtilis behavior has the greatest resemblance to what we report in D. discoideum. Upon starvation the population of B. subtilis partitions into sporulating and non-sporulating cells. Non-sporulating vegetative cells postpone their sporulation by consuming secondary metabolites and cannibalizing each other, and have the advantage of immediate growth upon arrival of nutrients9,46. In D. discoideum aggregation is required for sporulation. Since sporulation is beneficial only if the duration of starvation is long enough (Figure 6), and since cells cannot a priori sense the duration of starvation, population diversification should be the optimal response. This is exactly what we get with our model in Figure 6. We therefore propose that partitioning between non-aggregating and aggregating cells is a form of bet hedging in environments with unpredictable durations of starvation. Bet hedging behaviors result from epigenetic switching between different phenotypes. In D. discoideum, we show that a population of only aggregating (spores) or only non-aggregating cells re-partitions into aggregating and non-aggregating cell fates upon starvation following re-growth for a couple of cell divisions. This demonstrates the epigenetic bet hedging-like nature of population partitioning in D. discoideum.\n\nConsequently, our results have implications for studies of cooperation that use social amoebae as a model system. Studies on mixtures of non-isogenic cells show that some genetic clones bias their ratio into spores. Accordingly, clones associated with phenotypes enriched in the spore mass were qualified as cheaters, and phenotypes underrepresented in the spores as altruists47,48. However, the behavior of a mixture of more than two clones going through a series of growth and sporulation cycles cannot be entirely explained based on this ranking49. The whole life cycle needs to be taken into account, as competition occurs between strains not only during sporulation within aggregates but also at other steps such as unicellular growth, with complex trade-offs50,51. Here we characterize, in this respect, the aggregation step of the life cycle, and show that the previously neglected non-aggregating cell fraction constitutes a significant component of the population-level starvation response. This fraction is different for different genetic clones, it is at least of the order of the stalk cell subpopulation and interactions between clones do affect this fraction. Therefore, this additional unicellular cell fate needs to be taken into account when defining a clone’s behavior when alone and in mixtures. We propose to characterize amoebae behavior not only with respect to altruistic investment (spore vs. stalk in aggregates) but also with respect to social investment (aggregation vs. non-aggregation). This means that instead of classifying phenotypes as just altruistic and cheaters we may find a much richer repertoire, involving social cheaters (high aggregation efficiency but low investment into stalk), asocial altruists (low aggregation efficiency and high investment into stalk), asocial cheaters (low aggregation efficiency and low investment in the stalk) and so forth.\n\nPopulation partitioning can also be interpreted as probabilistic expression of social behavior. Genetic and non-genetic mechanisms regulate the probability of a cell acquiring a social/aggregating phenotype. It has been shown that such probabilistic expressions of social phenotype may be strong anti-cheating strategies and play an important role in stabilizing cooperation52,53. The results presented here reinforce the notion that one should allow individuals to 'opt out' of a social interaction to gain a more complete understanding, as has been argued for some time by game theoreticians54. For instance, allowing individuals to opt out of a social interaction may lead to evolutionary cycles52,55,56. Our results show that environmental stochasticity affecting relative fitness of social and asocial individuals may also favor opting out of at least a part of the population. It will be important to investigate further the role of population partitioning into aggregating/social and non-aggregating/asocial phenotypes on the stabilization of cooperation.\n\nOverall, we have demonstrated that the starvation stress response of the social amoebae Dictyostelium consists of the coexistence of a unicellular non-sporulating strategy and a multicellular sporulating strategy. We provide evidence that cell fate is determined by four types of factors: (i) autonomous, linked to cell genotype, (ii) environmental, (iii) dependent on gene × environment interactions, and (iv) dependent on cell-cell interactions. These social amoebae thus lie at the intersection of two key concepts in evolutionary microbiology, namely cooperation and bet hedging, and define a unique model system to explore this new frontier.\n\n\nData availability\n\nfigshare: Aggregation vs. nonaggregation strategies in Dictyostelium discoideum amoebae in response to starvation stress: raw data, doi: 10.6084/m9.figshare.105299757", "appendix": "Author contributions\n\n\n\nDD performed all microscopy experiments, cell culture and transformation, image analysis, statistical analysis, and conceived the mathematical model. MvB supervised the model formulation. CN produced the fluorescent reporter constructs, and conceived and set up the cell fraction quantification method. All authors contributed to the design of the study. All authors contributed to writing the manuscript and agree to publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nDD is the recipient of a PhD fellowship from the AXA Research Foundation at the Frontières du Vivant PhD program.\n\n\nAcknowledgements\n\nThe carA and pdsA mutants were kindly provided by Kerry Ann Sheppard at the Dicty Stock Center of Northwestern University in Chicago, and the phg2 mutant by Anna Marchetti and Pierre Cosson at the CMU in Geneva. We thank Vidyanand Nanjundiah, Santosh Sathe, Bahram Houchmandzadeh, Silvia de Monte, Sandrine Adiba, Madhu Priya, Pierre Cosson, Pierre Golstein, Dominique Schneider, and Nicolas Desprat for many helpful comments during the course of this work. We are indebted to Natale Scaramozzino for technical assistance, and to Shalmali Kamat during her internship at LIPhy Grenoble as well as Zak Frentz and Juliette BenArous at the Laboratory of Living Matter of Stanislas Leibler at Rockefeller University for their contributions during pilot experiments. We acknowledge Stanislas Leibler and all members of the Laboratory of Living Matter for many stimulating discussions and their invaluable support.\n\n\nSupplementary materials\n\n\n\n\nReferences\n\nStearns SC: The Evolutionary Significance of Phenotypic Plasticity. Bioscience. 1989; 39(7): 436–45. Publisher Full Text\n\nKussell E, Leibler S: Phenotypic diversity, population growth, and information in fluctuating environments. Science. 2005; 309(5743): 2075–8. PubMed Abstract \| Publisher Full Text\n\nAcar M, Mettetal JT, van Oudenaarden A: Stochastic switching as a survival strategy in fluctuating environments. Nat Genet. 2008; 40(4): 471–5. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDarja D, Minus VB, Clément N: Aggregation vs. nonaggregation strategies in Dictyostelium discoideum. amoebae in response to starvation stress: raw data. figshare. 2014. Data Source\n\nFrancis D, Eisenberg R: Genetic structure of natural populations of Dictyostelium discoideum, a cellular slime mold. Mol Ecol. 1993; 2(8): 385–391. PubMed Abstract \| Publisher Full Text" }	[ { "id": "5221", "date": "04 Jul 2014", "name": "Richard Gomer", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very nice explanation of why some Dictyostelium cells do not join into aggregates when the population of cells starves. Many of us have observed this phenomenon, and wondered why this happens. The explanation is that if nutrients suddenly appear while aggregated cells are undergoing development, the non-aggregating cells can immediately begin growth and proliferation, while the aggregated cells have to plod through development and then spore germination before they can start dividing. The authors show both data as well as nice mathematical models of this bet-hedging strategy.One minor correction - in Figure 1A 'loan' should be 'lawn'. In the future, finding the mechanism that causes a small percentage of cells to not aggregate may shed light into new mechanisms of cell population symmetry-breaking and differentiation.", "responses": [] }, { "id": "5848", "date": "18 Aug 2014", "name": "Paul Rainey", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an important paper that draws attention to non-aggregating cells of Dictyostelium and shows that under nutrient deprived conditions such cells arise by a stochastic mechanism and when considered alongside cells that commit to slug – and ultimately spore – formation appear to represent a bet-hedging strategy. Such a strategy may be adaptive in the face of uncertainty surrounding unpredictable fluctuations in resource availability. This little considered aspect of the biology of Dicty has significant implications for how we think about the function of this amoeba in its natural environment.The article content is of good quality and the experiments are well conducted. However, I suggest re-writing the section entitled “Genetics of population partitioning…” to bring greater clarity. Experiments showing that loners can switch back to spore-formers do not demonstrate that the switch is epigenetic (the switch could be mutational (a genetic switch)). Similarly, the hypothesis of bet hedging is not tested: it is an entirely reasonable hypothesis, but not proven.There is need to provide experimental details in the figure captions concerning number of replicates, nature of error and statistical analyses (where appropriate). Specific suggestionsAbstract“...studies of microbial cooperation...”I recommend deleting this. “Non-aggregating cells have an advantage over cells in aggregates since they resume growth earlier upon arrival of new nutrients, but have a shorter lifespan under prolonged starvation.”Compared to what? Compared to spores? “We find that phenotypic heterogeneities linked to cell nutritional state bias the representation of cells in the aggregating vs. non-aggregating fractions, and thus regulate population partitioning.”I probably wouldn't use the word regulate. Maybe just 'affect' “D.discoideum thus constitutes a model system lying at the intersection of microbial cooperation and bet hedging, defining a new frontier in microbiology and evolution studies”This is not such a great sentence. Rather than try and sell it in this way, I would recommend that the authors either delete the sentence, or emphasize that they have drawn attention to an overlooked aspect of the biology of Dicty that may have ecological relevance. Introduction“Yet, living environments typically deviate from these conditions.”Remove “living” “Our main motivation was to study a previously known but neglected fact that not all cells aggregate upon starvation.”Add reference Remove “While often considered an experimental error or just insignificant” “We asked whether the fraction of non-aggregating cells constitutes an important component of the adaptive response to stress.”I thought the main idea you are testing is the possibility that solitary cells constitute a bet hedging strategy that may have adaptive significance in the face of unpredictable changes in the nutritional status of the environment? Materials and methodsModel“The advantage that non-aggregating cells have is a head start when conditions improve, as spores produced by aggregating cells need time to develop.”The advantage may come even earlier than spore formation. Is the commitment to form fruit bodies terminal? If so, then right from the outset, there is likely to be an advantage for spreading risk. In many ways this is very similar to what has been worked out for Bacillus and the idea that the commitment to sporulation is delayed as long as possible (see work from Losick's group).Results“...the observation that non-aggregating cells are actively moving, live cells that are intermixed with aggregating cells at the onset of starvation”Remove the comma Phenotypic plasticity affects population partitioning“While stationary phase cells show no significant difference compared to exponentially growing cells, cells feeding on a homogenous bacterial lawn and thus gradually starving showed a 3-fold increase in the proportion of non-aggregating cells,6.3±3.17% (p=0.027).”Perhaps mention in the results what a homogeneous vs. heterogeneous lawn of bacteria means. “Four different media were tested: HL5 rich medium, FM minimal medium, NS with 85mM glucose (NS Glu) and NS medium.”Without going to the M&M I don't know how to read the differences between the media. Why did you not systematically change C / N and the ratio of C:N? “We conclude that nutritional state distinguishes non-aggregating cells from aggregating cells, and that interactions between cells according to their nutritional state biases further partitioning between aggregating and non-aggregation cell fates.”But what is it about the nutritional status of the media?Figure 2Axes – should Homog. and Heterog. lawn instead of loan. Legend - Mean and standard deviation of x replicates (state number of replicates). State the meaning of the lines. Also, was a posteriori test was applied (assuming the lines are indicating the result of this analysis). If this was ANOVA first and then a posteriori test, then state the ANOVA result as well (without this I don't know what the p levels mean).Figure 3Legend - See comments above regarding representation of statistical results.Genetics of population partitioning into aggregating and non-aggregating fractions This section is unclear to me. It is not what I would call \"genetics\". I am not sure to what extent this is an 'exploration of the genetic mechanisms affecting aggregating / non-agg fates'. I understand that the authors have taken two mutants that can be rescued when grown with wild type. I really don't understand what these experiments tell us. This should be clarified and made more explicit. Cell history and cell fateChange “...bet hedging-like strategy between...” to “stochastic switch affecting” “Answering this question allows us to: i) rule out any genetic differences between aggregating and non-aggregating cells and ii) examine the effect of epigenetic inheritance of cell fate.”This does not rule out the possibility of a genetic mechanism. For example, a genetic switch could be responsible. “This demonstrates the strong persistence of population partitioning and the fast loss of cell epigenetic memory.”Or a genetic switch. I think this shows that the solitary types are not mutants. I do not think this shows evidence for an epigenetic switch. Figure 4Legend - No. of replicates? Nature of error bar? etc.ModelThe model is useful and shows nicely that solitary cells and slug / spore forming cells have likely ecological relevance. One thing missing is any parameter to describe interactions among cells in the slugs / fruit body (and between different genotypes) that are likely to be important components of fitness, but perhaps at this stage there is insufficient empirical data for this to be usefully attempted. Figure 6I think you should make clear in the caption that these are results from a mathematical model.Discussion“We report that upon starvation stress a population of D. discoideum amoebae partitions...”I think you should avoid the term “stress”. It is meaningless. “For our detailed analysis of genetic and non-genetic contributions...”The analyses are not particularly “detailed”. I suggest removing this. “Bet hedging behaviors result from epigenetic switching between different phenotypes.”Not necessarily epigenetic. Take contingency loci in pathogenic bacteria for example. “This demonstrates the epigenetic bet hedging-like nature of population partitioning in D. discoideum.”No. It demonstrates neither epigenetic, nor that the strategy is a bet hedging one. The behaviour is consistent with a bet hedging strategy. “This means that instead of classifying phenotypes as just altruistic and cheaters we may find a much richer repertoire, involving social cheaters (high aggregation efficiency but low investment into stalk), asocial altruists (low aggregation efficiency and high investment into stalk), asocial cheaters (low aggregation efficiency and low investment in the stalk) and so forth.”I would suggest you could also put aside the anthropomorphic language and refer to solitary cells and slug / spore forming cells and their interactions. “Population partitioning can also be interpreted as probabilistic expression of social behavior.”See previous comment.", "responses": [] } ]	1	https://f1000research.com/articles/3-133
https://f1000research.com/articles/3-294/v1	04 Dec 14	{ "type": "Research Article", "title": "Characterization of the truncated hemoglobin THB1 from protein extracts of Chlamydomonas reinhardtii", "authors": [ "Eric A. Johnson", "Juliette T.J. Lecomte", "Eric A. Johnson" ], "abstract": "Truncated hemoglobins (TrHbs) belong to the hemoglobin superfamily, but unlike their distant vertebrate relatives, little is known about their principal physiologic functions. Several TrHbs have been studied in vitro using engineered recombinant peptides. These efforts have resulted in a wealth of knowledge about the chemical properties of TrHbs and have generated interesting functional leads. However, questions persist as to how closely these engineered proteins mimic their counterparts within the native cell. In this report, we examined THB1, one of several TrHbs from the model organism Chlamydomonas reinhardtii. The recombinant THB1 (rTHB1) has favorable solubility and stability properties and is an excellent candidate for in vitro characterization. Linking rTHB1 to the in vivo protein is a critical step in understanding the physiologic function of this protein. Using a simplified three-step purification protocol, 3.5-L batches of algal culture were processed to isolate 50–60 μL fractions enriched in THB1. These fractions of C. reinhardtii proteins were then subjected to physical examination. Using gel mobility, optical absorbance and immunoreactivity, THB1 was identified in these enriched fractions and its presence correlated with that of a heme molecule. Mass spectrometry confirmed this cofactor to be a type b heme and revealed that the native protein contains a co-translational modification consistent with amino-terminal acetylation following initial methionine cleavage.", "keywords": [ "Hemoglobins", "oxygen binding", "THB1", "Chlamydomonas reinhardtii" ], "content": "Introduction\n\nExtraordinary advances in structural biology and biothermodynamics are credited to vertebrate hemoglobins (Hb). This exemplar tetrameric assembly is a component of virtually all biochemistry textbooks, which emphasize its role in reversible molecular oxygen binding. Yet, the superfamily of Hbs is distributed in all domains of life, and phylogenetic analyses have traced its origins to the Archean eon, an era that preceded by billions of years the evolution of highly specialized proteins for the storage and delivery of dioxygen. In contrast to our detailed knowledge of Hbs from animals, little information is available about other Hbs; in fact, it can be said that the cellular functions of the majority of Hbs remain open to investigation.\n\nSequence data organize the Hb superfamily in three ancient lineages: M (myoglobin-like, containing the vertebrate Hbs), S (sensor-like), and T (truncated)1. The T lineage of interest to this report has representatives (TrHbs) in prokaryotes, fungi, and Viridiplantae1–3. Although many TrHb genes have been identified through genome sequencing, only a few have undergone biophysical examination at the protein level. Even fewer have been investigated within their native cell, in large part because of the difficulty in characterizing non-essential proteins present at low (nM) levels in organisms that cannot be cultured or are non-transformable. This “glaring lack of reliable information on function”4 contrasts with the ever-growing collection of sequences and prevents a complete understanding of the determinants of reactivity in Hbs. A considerable challenge in Hb research is deciphering how evolution has harnessed the helical folds of the M and S lineages (“3/3” helical arrangement) and the T lineage (“2/2” helical arrangement) to adapt to various environments and metabolic needs.\n\nMuch insight regarding TrHbs and their relationship to other Hbs has been based on experimentation using engineered, recombinant proteins. Although indispensable for an understanding of Hb chemistry, the use of recombinant material carries an important caveat. Unless the protein is extracted from the native cell, doubts should remain regarding the identity of the cofactor, if any, that is associated with the polypeptide in vivo. This is especially true for the proteins of photosynthetic organisms because a variety of hydrophobic chromophores may be available5 to occupy the heme cavity and perform functions independent of dioxygen binding. Co- and post-translational modifications of the polypeptide chain and the cofactor are also possible and can be assessed only with the native material. Thus, it is essential to inspect the properties of the native protein to validate the biophysical data.\n\nTHB1 is a TrHb from Chlamydomonas reinhardtii. This unicellular, diflagellate alga is a key model organism, used in diverse fields such as microbiology6, developmental biology7, photosynthesis8, optogenetics9, synthetic biology10 and biofuels11. In prior work, we prepared recombinant apoprotein based on the THB1 gene from C. reinhardtii. The holoprotein was then reconstituted with a ferric b heme and purified in vitro. This recombinant THB1 (rTHB1) remains a monomer even in concentrated (mM) solutions. Using mutagenesis, optical absorbance, and nuclear magnetic resonance spectroscopy, we identified the axial ligands to the heme iron as the proximal histidine and a distal lysine in both the ferric and ferrous states. We also determined that the distal lysine can be displaced by the diatomic molecules that are common ligands to other Hbs (O2, CO, NO•). In vitro, rTHB1 was found to possess the nitric oxide dioxygenase activity also exhibited by many Hbs12. Interestingly, in vivo studies performed in parallel with these recombinant studies linked the expression of THB1 to the NIT2 transcription factor13. This transcription factor is the only known positive acting regulatory factor for the induction of genes required for the nitrate assimilation pathway14. The inducible control of THB1 by NIT2 strongly suggests THB1 is also involved in nitrate metabolism.\n\nrTHB1 displays novel physico-chemical properties and is suitable for further analysis. The THB1 protein expressed in vivo has unique physiology and is present in an extensively characterized model organism. With these combined attributes, THB1 is an excellent protein to gain a deeper understanding of TrHbs. We describe here a traditional biochemical approach to the characterization of the protein expressed in its native environment with the goal to correlate the recombinant protein used in biophysical studies with the native protein.\n\n\nMaterials and methods\n\nAll chemicals used were scientific-grade and were purchased from Sigma-Aldrich unless otherwise noted.\n\nStrain CC-1690 was obtained through the Chlamydomonas Resource Center (University of Minnesota). Cells were maintained on Tris acetate phosphate (TAP) medium agar plates15 until use. Liquid cell cultures were grown in Sager-Granick M medium16,17, at 20°C under constant agitation, aeration with sterile air and illuminated with cool white fluorescent light on a 14/10 (on/off) cycle to synchronize cell growth.\n\nStrain CC-1690 (NIT1+, NIT2+, mt+) was grown in Sager-Granick M medium as described above until the culture reached a cell density of approximately 1–2 × 106 cells/mL in a volume of 3.5 L. Algal cells were harvested by centrifugation at 5,000 × g for 10 min at 4°C, then washed once with 20 mM Tris–HCl pH 8.0, 20 mM NaCl and again concentrated by centrifugation at 5,000 × g for 10 min at 4°C. The resulting cell pellet was immersed in liquid nitrogen and allowed to equilibrate for 5 min before transfer to ice for 5 min. The frozen pellet was thawed and resuspended in 20 mL of 20 mM Tris-HCl pH 8.0 at 4°C. After resuspension, the solution was again immersed in liquid nitrogen for 5 min and transferred to ice for 5 min. The solution was thawed to 4°C and immediately separated by centrifugation at 5,000 × g for 15 min. The supernatant was removed and separated by centrifugation at 20,000 × g for 30 min at 4°C, then at 30,000 × g for 30 min at 4°C. The supernatant, free of any visible cell debris, was flash-frozen in liquid nitrogen and stored at -80°C until further purified.\n\nThe 20 mL of protein suspension was diluted to 50 mL using 20 mM Tris-HCl pH 8.0, 20 mM NaCl, then passed over a 2 mL HiTrap Q Fast Flow column by fast protein liquid chromatography, FPLC, (GE Healthcare Life Sciences) using a flow rate of 1 mL/min. Following a brief wash with 20 mM Tris-HCl pH 8.0, 20 mM NaCl, protein was eluted from the column using a 20 mM–400 mM NaCl gradient run over the course of 1 h. Protein was collected from this gradient in 3-mL fractions and each fraction tested for the presence of THB1 by dot-blot immunodetection using custom-made rabbit polyclonal antibodies (Covance) raised against THB113. These antibodies were used at 1:10,000 dilution in TBS buffer with 5% milk. THB1 was found to elute from the column at approximately 100 mM NaCl. Positive fractions were pooled and concentrated to 2.5 mL using a 4-mL centrifugal concentrator with a 10,000 Da molecular weight cut-off. The resulting protein solution was separated on a 16/600 Superdex 75 column (GE Healthcare Life Sciences) by FPLC using a 1 mL/min flow rate. 1-mL fractions were collected and tested for THB1 using immunodetection as above. THB1 was found to elute from the column approximately 35 mL after the 40 mL void volume of the column. Positive fractions were pooled and again concentrated using a 4-mL centrifugal concentrator with a 10,000 Da molecular weight cut-off. The sample was concentrated to the stop volume of the concentrator, approximately 50–60 μL. The protein extract was stored at 4°C and used within 48 h.\n\nProtein samples were analyzed by native or denatured gel electrophoresis. Native electrophoresis used precast Any-kD TGX polyacrylamide gels (Bio-Rad) and 25 mM Tris, 192 mM glycine pH 8.3 in both the upper and lower reservoirs. The proteins were separated at 4°C and 100 V constant voltage for 2 h. Following the completion of the electrophoresis, the proteins were transferred to 0.45 μm nitrocellulose (Whatman) using a wet-tank transfer with 25 mM Tris, 192 mM glycine and 10% methanol as the transfer buffer. The transfer was performed at 4°C and constant amperage of 300 mA for 60 min. Following transfer, the nitrocellulose blot was washed once with distilled water. The presence of heme was detected by coating the surface of the nitrocellulose with a thin layer of ECL reagent (Immobilon Western Chemiluminescent HRP Substrate, Millipore) followed by imaging on a chemiluminescent imager (ProteinSimple). The nitrocellulose was then washed with water and the bound protein was detected using MemCode protein stain (ThermoScientific) as per manufacturer’s instructions. The blot was again imaged. The nitrocellulose was destained as per manufacturer’s instructions and blocked using TBS buffer with 5% milk for 30 min. The blot was then probed with polyclonal rabbit anti-THB1 antibodies at 1:10,000 dilution as described above and previously13.\n\nDenaturing gel electrophoresis was performed using precast 16.5% Tris-tricine (Bio-Rad) with 100 mM Tris, 100 mM Tricine and 0.1% SDS in both the upper and lower reservoirs. The voltage was a constant 100 V for 1.4 h. Proteins were then either stained with Silver Stain Plus (Bio-Rad) according to manufacturer’s instructions or transferred to nitrocellulose and used for immunostaining as described above with polyclonal antibodies against THB113, histone H3 (AbCam, Cambridge Massachusetts ab1791), nitrate reductase (Agrisera, Sweden AS08310) or β–subunit of the ATP synthase (Agrisera, Sweden AS03030).\n\nFollowing purification and concentration, a 10-μl sample was injected into a Waters Acquity/Xevo-G2 UPLC-MS and analyzed using reverse-phase chromatography on a Waters BEH C4 (2.1 mm × 50) column (300 Å, 1.7 µm resin) using a mix of water/acetonitrile (in 0.1% formic acid) over a 10 min separation period. The solvent profile was 0% acetonitrile, ACN, (0–1 min), gradient to 80% ACN (1–7.5 min), hold at 80% ACN (7.5–8.5 min) and 0% ACN (8.5–10 min). Following chromatography, the sample was immediately injected into the QTof MS/MS mass spectrometer. A locked mass was used to calibrate found masses within the sample using the intermittent injection of a known sample of leu-enkephalin (Mr 556.2771) continuously throughout the analysis. The data collected during the analysis were processed using the BioPharmaLynx software package (Waters).\n\nFollowing the purification and concentration described above, a 2 μL sample was placed onto a NanoDrop 2000c spectrometer (ThermoScientific) and spectra obtained according to manufacturer’s instructions.\n\n\nResults\n\nThe unicellular green alga C. reinhardtii contains approximately 140 mg of total protein per 1 g cells15. Previous work has identified THB1 within these cells, but both immunodetection results and qPCR of gene transcripts suggest that THB1 is not an abundant cellular component. We determined that the THB1 gene is regulated by the NIT2 gene for nitrogen metabolism13, and therefore for these experiments selected the wild-type strain of C. reinhardtii (CC-1690 also called 21 gr) known to contain the NIT2 gene (unlike the common laboratory strains CC-124 or CC-125 also called 137c). Owing to this low induced protein expression, isolation of the protein from whole cells of C. reinhardtii requires dramatic enrichment of THB1 and exclusion of the vast majority of the cell’s other proteins. The approach taken in this report comprises three steps that avoid the use of detergent and enrich progressively a fraction of the total cell protein with THB1. Although total purity was not achieved by the method, enough surrounding protein could be removed so that physical aspects of THB1 were identified within the pool of remaining proteins.\n\nThe first enrichment step involves the freeze fracturing of whole cells. Following low-speed sedimentation, cell pellets are flash-frozen in liquid nitrogen that effectively ruptures the cell without severely disrupting its membranes. Following rupture, soluble proteins partition to the lysate, while proteins bound to membranes or within larger organelles are removed through a series of centrifugation steps (Figure 1A). This facilitates the release of the soluble proteins while retaining, and disposing of, the majority of the contents within the cell body. This gentle procedure also leaves pigmented complexes predominantly associated with larger membrane structures that do not partition to the lysate. As THB1 does partition to the lysate, this result suggests that in the cell, THB1 is a soluble protein similar to its recombinant counterpart (see introduction). Following the isolation of the lysate, the soluble cellular proteins are further enriched by anion-exchange chromatography (Figure 1B, lane 3). Previous work with rTHB1 has shown that the protein tends to bind weakly to anion-exchange resins13. When the soluble cellular proteins were passed over the resin, THB1 did bind but eluted early when a salt gradient was applied to the column. Further enrichment involved size-exclusion chromatography, which both enriched THB1 (Figure 1C, lane 4) and excluded other proteins with molecular weights substantially larger than that of THB1 (Figure 1B, lane 4).\n\nProtein samples from different stages of purification were analyzed by electrophoresis. (A) Samples of protein extracts before and after lysis of C. reinhardtii cells with liquid nitrogen. Proteins separated on 16.5% Tris-tricine gel then transferred to nitrocellulose and immunostained with antibodies against proteins localized to different cellular compartments. Lysis by liquid nitrogen enriches the lysate with soluble proteins without enrichment of proteins found in major algal organelles. Histone H3 protein is located in the nucleus, nitrate reductase is a soluble cytosolic protein and the ATP synthase β subunit is part of the thylakoid membrane of the chloroplast. (B) Samples from different steps in the purification procedure were separated by electrophoresis. Following separation, the proteins within the gel were visualized using silver stain. (C) Proteins prepared identically to those detected in panel B were transferred to nitrocellulose followed by immunostaining with polyclonal antibodies specific for THB1. In addition to the protein samples, a lane was used for molecular weight markers (Spectra LR, ThermoScientific). The numbers indicated between the panels represents location of the markers (kDa molecular mass). WC, whole cell protein extract. Lys, protein extract lysate, and Pel, protein pellet following liquid nitrogen fracturing. QFF, concentrated sample following anion exchange chromatography. S75, concentrated sample following separation on the Superdex 75 column.\n\nThe protein fraction following Superdex 75 reduced an initial volume of 3.5 L of cells to yield approximately 50–60 μL of isolated proteins. The protein solution was visibly light pink in color, and its optical spectrum (Figure 2) showed a distinct absorption maximum corresponding to the known Soret band for ferric THB1 (410 nm). Using the estimated extinction coefficient for this form of THB113, it can be estimated that the solution was approximately 5 μM in THB1.\n\nA 2-μL drop of the concentrated fraction following separation on Superdex 75 was scanned using a nanodrop spectrometer (average of four scans). Maximum absorbance occurs at 410 nm and corresponds to the Soret peak of rTHB1 at neutral pH in the ferric state13. Based upon the published extinction coefficient for the recombinant protein under these conditions, this sample contains approximately 5 μM THB1. Note that the presence of ferric rather than ferrous protein may be the result of oxidation during the purification procedure. Q bands (500–600 nm) are not detected because of the low signal-to-noise ratio.\n\nThe absorbance spectrum of the solution matched that of a heme protein. However, a direct link of this pigment and THB1 is not established since the solution still contains other proteins in addition to THB1. To investigate the colocalization of THB1 with any bound cofactor, native electrophoresis can be used, as tightly bound cofactors will remain associated with the protein while it migrates through the gel. Following Superdex 75 chromatography the proteins in the resulting solution were separated using native gel conditions and then transferred to nitrocellulose. Protein staining (Figure 3A) shows that the various components of the samples were separated within the native gel. Chemiluminescence “heme stain”18,19 (Figure 3B) displays the presence of a single band. Following protein staining and heme staining, the same nitrocellulose blot can again be used for immunostaining with polyclonal antibodies against THB1 (Figure 3C). Immunostaining is much more sensitive than heme staining, and very short exposure times were sufficient to detect stained bands on the nitrocellulose. The procedure revealed a major band at the same position as the heme signal was obtained. In addition, rTHB1 (with known heme content) reacts with both the heme stain and the immunostain, although when electrophoresis was performed under native conditions, the recombinant THB1 had a slightly lower mobility in the gel than the native protein.\n\nFollowing separation on the Superdex 75 column and concentration, the sample (identical to the sample used in Figure 2) was separated on an acrylamide gel under native conditions and transferred to nitrocellulose. The same nitrocellulose was stained to detect (A) total protein, (B) heme and (C) THB1 as described in the methods section. Dilutions of the sample were used owing to differing sensitivities of the different detection methods with immunodetection being much more sensitive than the heme or protein stain. The immunodetection using anti-THB1 antibodies therefore has high background even at very short exposure times; however, only a single protein band co-localizes with both heme stain and THB1 supporting that THB1 is associated with heme. Quantities are marked in pmol above each lane.\n\nThe components of the Superdex 75 fraction were investigated using mass spectrometry. To establish a protocol, the procedure was first performed with the recombinant protein. rTHB1 elutes from the C4 column at approximately 4.9 min using the conditions described in the methods section. The spectrum of the recombinant protein, displayed as the ratio of relative mass to charge number (m/z) (Figure 4A), shows an m/z distribution for a protein and a single strong m/z peak at 616. This single m/z signal is consistent with that of the b heme (molecular mass 616 Da) bound to the recombinant THB1. The relative mass of the protein (apo rTHB1) is 14564 (Figure 4B), as expected if the initial methionine is cleaved.\n\nA 10-μL sample was separated on a C4 column and detected as intact protein by mass spectrometry as described in the methods section. (A) The unprocessed mass spectrum of rTHB1 as represented by the relative signal intensity at each mass-to-charge value between 400 and 2000. (B) The same data used to generate the spectrum in panel A were subject to deconvolution using the BioPharmaLynx software program to determine the mass of intact proteins within the sample. (C) The unprocessed mass spectrum (as in panel A) of the concentrated sample from C. reinhardtii following separation on the Superdex 75 column. (D) The data shown in panel C were subjected to processing as in panel B. panels B and D were processed under the same conditions.\n\nWhen the mass analysis was repeated on the purified proteins from C. reinhardtii the m/z spectrum was complicated by the presence of additional proteins (Figure 4C). One notable similarity between the two samples is the strong, single signal at m/z = 616. This suggests that a molecule that exhibits the same m/z as the b heme in Figure 4A is among the purified algal proteins, further supporting the hypothesis that this sample contains b heme bound within the native THB1. Deconvolution of the purified protein spectrum (Figure 4D) reveals a group of molecules whose distribution of molecular mass roughly correlates to the distribution of protein bands seen in the silver-stained denatured protein electrophoretic gel (Figure 1B). Examination of these individual relative masses failed to find any with a value of 14564 corresponding to the recombinant THB1. There was, however, a significant signal at a mass of 14606, which represents an increase of 42 above the rTHB1 relative mass. We concluded that the 14606 signal did correspond to THB1, modified by the acetylation of a single functional group, which replaces a hydrogen atom by a COCH3 moiety. We attribute the slightly different migrations of rTHB1 and THB1 in the gel of Figure 3C to this modification of the native protein.\n\nIn previous work, THB1 was found within the soluble protein extracts of C. reinhardtii flagella20. The protein was identified by excision from polyacrylamide gels, followed by protease digestion and peptide mass spectrometry. Two peptides from this extracted protein were attributed to THB1, one of them encompassing the 13 N-terminal residues of the protein. Re-examination of the peptide mass spectrometry data showed the assignment of N-terminal acetylation to this fragment (George Witman, personal communication). In light of this additional information, it seems most likely that the increased molecular weight seen in native form of THB1 comes from acetylation of the N-terminal peptide, in this case an alanine (Ala2). In sum, the mass spectral data obtained on the intact protein indicate that THB1 is present in the C. reinhardtii protein extract, that it lacks its initial methionine, has N-terminal acetylation, and that a b heme is associated with the protein.\n\nIn vitro experiments have shown that TrHbs (and many Hbs in general) are capable of processing reactive nitrogen species to less toxic molecules. This activity has been specifically shown in rTHB113, which converts nitric oxide to nitrate via the nitric oxide dioxygenase (NOD) reaction12. As an additional assessment of the properties of the native protein, we attempted to assay this activity on material generated after passage through the Superdex 75 column. The small sample volumes complicated measurement of NOD activity and necessitated the use of microliter spectrophotometry. Given these limitations, it was not possible to determine accurate yields of nitric oxide conversion. The results of these attempts, although not presented because of high background and low number of replicates, did not conflict with the high yields observed with rTHB113, and therefore NOD activity is still a plausible role for THB1 in vivo.\n\n\nDiscussion\n\nTo date hundreds of TrHb genes have been identified within the genomes of bacteria, fungi, plants and algae1,2. Despite the accumulation of sequences and remarkable progress in genetic and structural knowledge, there is limited information about the physiological role of these proteins within most organisms. In some photosynthetic prokaryotes, there is evidence that constitutive expression of TrHbs helps the cell survive exposure to reactive oxygen species (ROS)21 or reactive nitrogen species (RNS)22. The connection between TrHbs and ROS/RNS mediation has also been identified within non-photosynthetic organisms such as Mycobacterium tuberculosis, an organism in which TrHbs are linked to protection of the cell from the cytotoxic effects of external NO•23,24. The cytotoxic effects of internal NO• maybe mitigated in some plants and algae where TrHbs are induced under hypoxic stress25–27. The study of TrHbs therefore helps our understanding of both intracellular stress and intercellular “redox warfare”28. Ideally TrHbs may be targets for the design of synthetic molecules manufactured to control these events, for therapeutic benefit in humans or economic benefit in plants and algae.\n\nTHB1 from C. reinhardtii provides a welcome opportunity to expand the examination of the TrHb lineage – THB1 is present in a very well characterized organism, the native protein is linked to a critical metabolic pathway involving RNS, and the recombinant protein is amenable to detailed in vitro studies. These in vitro studies are essential because properties that dictate chemistry, such as the identity of the distal ligand to the heme iron, the ability to bind exogenous molecules, and the iron redox potential, cannot be inferred from the amino acid sequence and are best determined using large quantities of pure material with and without isotopic labels or amino acid replacements. However, in order to extrapolate in vitro findings to in vivo conditions, it is imperative to question how well the recombinant wild-type model mimics the native protein. In the experiments presented here, we have sought to bridge the in vitro and in vivo studies by comparing the physical characteristics of rTHB1 to those of the protein from the native cell.\n\nOur study revealed one difference between the THB1 polypeptides synthesized by E. coli and C. reinhardtii. In many eukaryotic cells the majority of cytoplasmic proteins are N-terminally acetylated29 for purposes that are not entirely understood30. N-terminal (Nt) acetylation has also been observed in C. reinhardtii, though studies have focused on chloroplast targeted proteins31 with the conclusion that the modification may retard degradation. We have found that THB1 is acetylated, most likely at the N-terminus. Whether this modification contributes to the stability of cytosolic proteins such as THB1 is not known. The first two eukaryotic TrHbs to be isolated from their native organisms (the ciliates Paramecium caudatum and Tetrahymena pyriformis) exhibit a blocked N-terminus when subjected to amino acid analysis. The blocking group was identified as an acetyl moiety in P. caudatum globin32 and inferred in T. pyriformis globin33. Thus, of the three native eukaryotic TrHbs peptides so far characterized, all three appear to contain the same modification, though its consequences remain to be determined.\n\nThe three-dimensional structure of several relatives of THB1 has been solved. These include globin domains from P. caudatum (PDB ID 1DLW), two cyanobacterial species (Synechocystis sp. PCC 6803, PDB ID 1RTX and Synechococcus sp. PCC 7002, PDB ID 4MAX) and Chlamydomonas eugametos (PDB ID 1DLY). Nuclear magnetic resonance (NMR) data collected on rTHB113 indicate that the heme domain is structurally similar to other characterized TrHbs. Structural data (NMR and X-ray crystallography, Matthew Preimesberger and Selena Rice, personal communication) also concur that the N-terminal extension is disordered (data not shown). This leads us to assume that acetylation has minimal impact on the reactivity and enzymatic properties of the native protein relative to its recombinant counterpart.\n\nSimilarity between recombinant and native THB1 proteins extends to the holoprotein forms. The nature of bound cofactors is a critical feature of any enzyme. For hemoglobins, the “standard” cofactor is a b heme (Fe-protoporphyrin IX), but instances of covalent heme attachment have been reported in Synechocystis34 and Synechococcus22 TrHbs. This or other types of post-translational modification may occur in TrHbs and should not be overlooked. More importantly, many derivatives of the tetrapyrrole core and other large hydrophobic molecules are present in living cells, especially photosynthetic ones. The 3/3 globin fold is known to accommodate several such molecules, for example chlorophyllin35, chlorophyllide36, and open tetrapyrroles37–39, and the same might be expected of the 2/2 proteins. Our results establish that native THB1, as extracted from C. reinhardtii cells by gentle methods, contains an unmodified b heme as was incorporated into the recombinant apoprotein to generate rTHB1. In addition, no significant quantity of a species with mass indicating covalent attachment of the heme was detected. It must also be noted that although we isolated holo THB1, we cannot rule out the possibility that some amount of (functional) apoprotein is present or can exist under different growth conditions. Further examination of the protein within the cell would be required to address this question.\n\nThe results presented here compared the recombinant and native versions of the THB1 protein from C. reinhardtii. Both proteins exhibit similar optical properties; both are recognized by the same antibody, exhibit similar migration upon gel electrophoresis and possess the same cofactor. Co-translational modification (acetylation) occurs to the native protein, but the location of the modification in the three-dimensional structure suggests the modification’s role (if any) is regulatory and does not impact the protein’s activity. Taken together, the data reinforce the use of rTHB1 as a model for the native protein within C. reinhardtii. Future work will continue to utilize in vitro and in vivo experiments with the goal of understanding the activity of TrHbs from both the molecular and physiologic perspectives.\n\n\nData availability\n\nF1000Research: Dataset 1. Mass spectrometry data of purified proteins, 10.5256/f1000research.5873.d3988440", "appendix": "Author contributions\n\n\n\nEAJ and JTJL conceived the study. EAJ designed and executed the experiments. EAJ prepared the first draft of the manuscript. EAJ and JTJL revised draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the National Science Foundation under grant number MCB-1330488.\n\n\nAcknowledgements\n\nThe authors wish to thank Dr. Greg Bowman for the use of his chromatographic equipment, Dr. Phil Mortimer for his technical assistance, and the Johns Hopkins University Mass Spectrometry Facility. 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Data Source" }	[ { "id": "6905", "date": "16 Dec 2014", "name": "Michael Berenbrink", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a solid study, addressing an important point in the study of truncated haemoglobins (and indeed all proteins), namely the verification of results obtained on recombinant proteins also with proteins obtained in the native state. The experimental design and methods are appropriate, the results are clearly presented and carefully discussed, and the conclusions are justified and supported by the data.", "responses": [] }, { "id": "6903", "date": "16 Dec 2014", "name": "Cinzia Verde", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn their Research article \"Characterization of the truncated hemoglobin THB1 from proteinextracts of Chlamydomonas reinhardtii\", Johnson & Lecomte describe the structural properties of a truncated globin, purified by traditional biochemical approaches, from the extracts of Chlamydomonas reinhardtii with the aim to correlate the recombinant protein previously studied by biophysical techniques with the protein expressed in its native environment.The report is comprehensive and up to date and will certainly become a key reference in the field. The topic is of interest to many readers and to my knowledge has not been covered by many articles. This article highlights the similarities and differences between recombinant protein over-expressed in Escherichia coli and native protein. Although extensive research has been conducted to investigate the physiological function of truncated globins, knowledge of their functional role is limited by the lack of facile methods for in vivo studies. Consequently, the most telling insights into the physiological role of these proteins have arisen via classical approach on the recombinant protein. As the authors note, potential correlation between recombinant and native proteins may help to formulate plausible mechanisms for the function of these globins, and this is a particularly interesting highlight of this work. The paper is short but very well written, with a balanced view of the literature and makes for interesting reading. I have no comments to offer as to how to improve the paper and recommend indexing as is.", "responses": [] }, { "id": "6902", "date": "31 Dec 2014", "name": "Thomas Gorr", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nUnraveling the functional properties of truncated hemoglobins (TrHBs) is both important and revealing as these mini globins of todays cyanobacteria and green algae not only function on the basis of a simplified two-on-two helical topology (in contrast to the three-on-three helices of canonical globins) but also link back to primordial globins that first appeared some 3 billion years ago in a biosphere virtually free of oxygen. Perhaps in reminiscence of this ancestry, many TrHBs function indeed in roles related to reactive nitrogen usage rather than as dioxygen sensing and/or transporting heme-binding proteins. A previous publication by the same authors (i.e. Johnson et al., 2014) documented efficient NO• dioxygenase activity along with proximal histidine and displaceable distal lysine heme ligands for the recombinant THB1 (rTHB1) truncated hemoglobin of the unicellular algae Chlamydomonas reinhardtii. However, to further our functional understanding of TrHBs it is important to elicit how closely recombinant proteins mimic their counterparts within the native cell. Direct investigations of native TrHBs are rendered difficult due to low level (nM range) expression of these proteins and the fact that many of these organisms can neither be cultured nor transformed. To address this issue, the current publication by Drs. Johnson and Lecomte aims to link biochemical properties of Clamydomonas rTHB1 to the in vivo protein. By using a straightforward 3-step purification protocol including freeze-thaw separation of soluble proteins followed by anion-exchange and Superdex size-exclusion FPLC chromatography steps, the authors were able to enrich native THB1 to micromolar concentrations, as judged by light absorption of the 410nm Sorret band for ferric THB1. Native gel electrophoresis and chemiluminescent ECL staining of the final Superdex purification fraction confirmed presence of a single heme-bound protein band that was positively identified as THB1 by subsequent immunostaining with self-generated polyclonal anti-THB1 antibodies (Johnson et al. Biochemistry 2014, 53, 4573-4589). Recombinant THB1 used as electrophoretical reference revealed a slightly lower mobility than the native globin. Presence of a 616 Da b heme (Fe-protoporphyrin IX), lack of the initial methionine and N-terminal acetylation at Ala2 was all demonstrated for the native protein (Mw=14606 Da) by UPLC-MS, again in comparison with rTHB1. As expected, the recombinant protein (Mw=14564 Da; ΔMw (native-recombinant)=42 Da; corresponds to mass increase by COCH3 moiety in replacement of one hydrogen atom) was devoid of any post-translational modification (acetylation). This missing acetyl moiety in rTHB1 might also explain the slightly different electrophoretic mobility in native gels. An N-terminal amino acid sequence, blocked for Edman degradation by an acetyl moiety, has also been detected or inferred for TrHBs of the clitated protozoans Paramecium caudatum and Tetrahymena pyroformis. Thus, of the three native eukaryotic TrHbs peptides so far characterized, all three appear to contain the same modification, though its consequences remain to be determined. Based on unpublished NMR and X-ray crystallography results by Drs. Preimesberger and Rice (pers. communication), the N-terminal extension, however, appears to be disordered and of minimal impact on reactivity and enzymatic properties in TrHbs. Together, this publications contains an easy-to-implement protocol for the purification of low level expressed native TrHbs and, as such, represents an important contribution for further links of the biochemical properties of recombinant and native truncated hemoglobins.", "responses": [] }, { "id": "6904", "date": "12 Jan 2015", "name": "Suman Kundu", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nExpanding genome sequence data has led to the discovery of a plethora of hemoglobins from diverse organisms through bioinformatics analysis of signature sequences. The verification of putative hemoglobins from such genomes, however, came only from investigation of recombinant hemoglobins in heterologous system. For way too long we have dabbled in understanding of biochemical, kinetic and structural properties of such recombinant hemoglobins. While such investigations have unraveled key insight and unique features of the novel hemoglobins with hypothesis for putative functions in vivo, a final word on their function is still awaited. The non-essentiality of these genes, their low physiological concentrations and lack of obvious relations to phenotypes has also kept an insight into their functional roles at bay from occasional in vivo genetic studies. A potential source of further insight into these proteins might thus lie in their post-translational modifications, modifications of the heme or their ability to interact with other key proteins in vivo, thus playing key roles at the proteome level. The investigation by Johnson and Lecomte addresses this key issue by characterization of an algal hemoglobin from native source in comparison to its recombinant counterpart. The experiments have been beautifully executed and the conclusions carefully laid down. While several properties of the native protein are indeed similar to the recombinant version, the N-terminal acetylation of the native hemoglobin indeed reveal new opportunities in understanding the function of these classes of proteins. It would have been exciting to see similarities or differences in the kinetic properties (on rates, off rates and ligand binding affinities) of the recombinant and native algal hemoglobins, which can provide additional information into their ligand-dependent functional roles. I am sure that the authors themselves or some other group will investigate this aspect as well in the near future. I am hopeful that this pioneering investigation will inspire other similar studies taking us into yet unknown territories of hemoglobin features and functions.", "responses": [] } ]	1	https://f1000research.com/articles/3-294
https://f1000research.com/articles/3-179/v1	30 Jul 14	{ "type": "Opinion Article", "title": "Why are neurotransmitters neurotoxic? An evolutionary perspective", "authors": [ "Keith D. Harris", "Meital Weiss", "Amotz Zahavi", "Meital Weiss", "Amotz Zahavi" ], "abstract": "In the CNS, minor changes in the concentration of neurotransmitters such as glutamate or dopamine can lead to neurodegenerative diseases. We present an evolutionary perspective on the function of neurotransmitter toxicity in the CNS. We hypothesize that neurotransmitters are selected because of their toxicity, which serves as a test of neuron quality and facilitates the selection of neuronal pathways. This perspective may offer additional explanations for the reduction of neurotransmitter concentration in the CNS with age, and suggest an additional role for the blood-brain barrier. It may also suggest a connection between the specific toxicity of the neurotransmitters released in a specific region of the CNS, and elucidate their role as chemicals that are optimal for testing the quality of cells in that region.", "keywords": [ "Some non-peptide chemicals that function as neurotransmitters in the central nervous system (CNS)", "such as dopamine and serotonin", "have toxic effects1–4. Neurodegeneration can result from the deregulation of the concentration of these neurotransmitters5–7. It is known that neurotransmitters such as serotonin", "acetylcholine (ACh)", "glutamate and gamma-aminobutyric acid (GABA) function as signals between non neuronal cells in the periphery8–12", "and have evolutionarily conserved roles", "serving also as signals in plants13", "14 and unicellular organisms15. This does not necessarily explain their adaptive role as signals in the CNS", "as at synapses a variety of less toxic chemicals could have served the same role", "had they been loaded into vesicles in the pre-synaptic neuron and had complementary receptors on the post-synaptic neuron. In the following we attempt to highlight the potential insights that may arise from applying the theory of signal selection16 to the evolution of signals between cells in multicellular organisms. The theory of signal selection", "based on the handicap principle", "suggests that the properties of the signal serve as a test of the information encoded in the signal. The theory revolutionized the study of signaling between organisms17", "18. The application of the theory to the evolution of neurotransmitters suggests that neurotransmitters are selected in part because of their toxicity", "which serves as a test of the quality of the releasing cell and its connectivity with neighboring cells", "and facilitates the selection of neuronal pathways." ], "content": "Introduction\n\nSome non-peptide chemicals that function as neurotransmitters in the central nervous system (CNS), such as dopamine and serotonin, have toxic effects1–4. Neurodegeneration can result from the deregulation of the concentration of these neurotransmitters5–7. It is known that neurotransmitters such as serotonin, acetylcholine (ACh), glutamate and gamma-aminobutyric acid (GABA) function as signals between non neuronal cells in the periphery8–12, and have evolutionarily conserved roles, serving also as signals in plants13,14 and unicellular organisms15. This does not necessarily explain their adaptive role as signals in the CNS, as at synapses a variety of less toxic chemicals could have served the same role, had they been loaded into vesicles in the pre-synaptic neuron and had complementary receptors on the post-synaptic neuron. In the following we attempt to highlight the potential insights that may arise from applying the theory of signal selection16 to the evolution of signals between cells in multicellular organisms. The theory of signal selection, based on the handicap principle, suggests that the properties of the signal serve as a test of the information encoded in the signal. The theory revolutionized the study of signaling between organisms17,18. The application of the theory to the evolution of neurotransmitters suggests that neurotransmitters are selected in part because of their toxicity, which serves as a test of the quality of the releasing cell and its connectivity with neighboring cells, and facilitates the selection of neuronal pathways.\n\n\nThe theory of signal selection\n\nThe theory of signal selection was developed by Zahavi19,20 to explain why peahens are stimulated by a trait that imposes a handicap on the male, rather than paying attention to more positive traits in the males that court them. Zahavi suggested that peahens are attracted by peacocks that carry the burden of a long and heavy tail because this burden constitutes a handicap that tests the quality of the displaying peacock. This interpretation pointed at the objective information provided by the signal, which results in the peahen responding to one peacock and rejecting others; it is not coincidental that peahens are attracted to males with heavy tails, rather, it is the tested and reliable information provided by the cumbersome tail that selected for the interest of the female in the level of the handicap imposed on the male by its tail.\n\nWe suggest that, similarly to the burden imposed by the peacock’s tail, toxicity is necessary to impose a specific chemical burden on the signaling cell to ensure that the signal inherently provides reliable information on some properties of the signaling cell. It is reasonable to assume that if signals within multicellular organisms were consensus signals that did not inherently correlate to a specific metabolic activity of the signaling cell, a larger variety of chemicals could have been selected as signals within multicellular organisms. In addition, phenotypes which had not developed to signal could signal in error, while the level of the signal could misrepresent the metabolic state of the signaler. We suggest that the investment in reliable signaling in multicellular organisms is necessary in order to reduce the potential harm of such errors16. Tests must be difficult in order to provide meaningful and reliable results16, and hence we expect that, if neurotransmitters also test the quality of the releasing cell, they should be directly toxic in a way that tests the message encoded in the signal.\n\n\nNeurotransmitter toxicity and its implication in neurodegeneration\n\nIn the CNS, neurotransmitters play a central role in relaying information at chemical synapses. This role involves their vesicular secretion by the pre-synaptic cell and interaction with receptors on the post-synaptic cell. However, neurotransmitters are also released outside synapses in high concentrations prior to blood-brain barrier development21,22 and as part of non-synaptic forms of intercellular communication in the mature brain23. Synaptic transmission requires the rapid clearance of the secreted or released neurotransmitter via uptake by neurons and astrocytes24. When these mechanisms are deregulated, the accumulation of neurotransmitter in the extracellular matrix can lead to neurodegeneration5–7. Here we review briefly the toxicity of some neurotransmitters and its role in neurodegeneration.\n\n\nGlutamate\n\nGlutamate exerts neurotoxicity via excitotoxicity caused by the overactivation of NMDA receptors25 and oxidative toxicity caused by the inhibition of cysteine uptake via uptake by the cysteine-glutamate anti-porter26. As glutamate uptake is an energy-dependent process that involves the co-transport of sodium27, glutamate uptake is reversed in hypoxic conditions and leads to an increase in extracellular glutamate28. The increase of extracellular glutamate has been implicated as a causative factor in numerous pathologies, including stroke29, Huntington’s disease, Parkinson’s disease and amyotrophic lateral sclerosis30.\n\nDespite its abundance, glutamate is stored mostly in subcellular compartments31: in astrocytes its uptake is coupled with its conversion to glutamine32 and in neurons the synthesis of glutamate from 2-oxoglutarate33 or glutamine32 is correlated to its uptake into vesicles, suggesting that it is also potentially toxic within the cytoplasm. In addition to glutamate toxicity that is mediated by its interaction with receptors and secondary to its uptake mechanisms, evidence of the interaction of glutamate with oxygen radicals could point to potential direct damage of glutamate to membranes. In the presence of hydroxyl radicals and molecular oxygen, glutamate is oxidized to 2-oxoglutarate in a reaction that releases hydrogen peroxide34,35. Glutamate in particular has a relatively high yield of peroxide in the presence of oxygen radicals, relative to glutamine, glycine and aspartate34. This process is also iron-dependent, the presence of which is a causative factor of neurodegeneration involving radical oxygen species36.\n\n\nDopamine\n\nDopamine is involved in the pathogenesis of Parkinson’s disease, which involves the degeneration of dopaminergic neurons in the substantia nigra, leading to motor dysfunction5,6. The loss of dopaminergic neurons has been linked to dopamine’s cytotoxicity that results from the deregulation of its metabolism in these neurons6.\n\nDopamine is directly toxic in its oxidized semiquinone and quinone forms1,37. Dopamine toxicity is also related to the presence of metal ions such as iron4, which increase its oxidation to neurotoxic metabolites38, while metal ion chelators have a protective effect in Parkinson’s disease39. It has already been suggested that redox mechanisms that render intracellular dopamine toxic in the cytosol could also render extracellular dopamine toxic3.\n\n\nSerotonin\n\nSerotonin is sensitive to oxygen radicals, and its indole moiety is readily oxidized in the presence of hydroxyl radicals to form neurotoxic metabolites of serotonin1. The indole moiety of serotonin can undergo oxidation by indoleamine 2,3-dioxygenase to form kynurenine, which can be metabolized further into various neurotoxic chemicals40. This pathway of serotonin metabolism has been implicated in neurodegeneration associated with depression41. Serotonin is toxic in the presence of copper42, causing intracellular damage such as DNA strand cleavage43. Serotonin is also toxic in the presence of iron2, causing mitochondrial damage44. This suggests a role for serotonin in copper and iron mediated neurodegeneration.\n\nSerotonin can also interact with lipid membranes45, partially intercalating into the phospholipid layer and thus causing structural changes in the membrane. It has been shown that the interaction of neurotransmitters with the cell membrane can have a non-specific anesthetic effect on receptor activity46, and so chronic exposure to serotonin may alter membranal homeostasis.\n\n\nAcetylcholine\n\nAs far as we are aware, there is currently no experimental evidence of direct ACh toxicity. However, the overstimulation of ACh receptors as a result of ACh accumulation that is caused by acetylcholinesterase inhibition can lead to cholinergic toxicity47,48. This toxicity may involve the release of choline from phosphatidylcholine that is downstream of muscarinic ACh receptors49, leading to phosphatidylcholine depletion. In addition, the use of nicotinic ACh receptor antagonists has shown to reduce the neurotoxicity of the Alzheimer’s disease-related peptide, β-amyloid50.\n\nACh interacts with lipid bilayers and elicits changes in the organization of the lipid bilayer51. This interaction is non-specific, slower than receptor activation, and has a longer duration46. We speculate that the accumulation of ACh could interfere with the membrane morphology46 and consequently may interfere with its function.\n\n\nThe function of neurotransmitters in the brain – some considerations resulting from our evolutionary perspective\n\nThe consideration of a function for neurotransmitters as a reliable representation of the specific activity of the releasing cell, rather than simply as chemicals that facilitate the transfer of information between neurons, may contribute novel deliberations and interpretations of known phenomena.\n\nThe formation of connections between neurons in the vertebrate CNS during embryogenesis and development is a dynamic process in which neurons that do not form synapses are eliminated52,53, while neurons forming new synapses survive into adulthood54,55. In addition, since neurons have an array of potential connections, a selection process is involved in the development and ongoing activity of neuronal networks52,55–57. Hence, we suggest that the toxic neurotransmitters that are released from neurons in the CNS function as tests of neuronal quality. The toxicity is important for the process of selection that is involved the selection of the optimal pathways for relaying information between and within specialized CNS centers.\n\nA better reflection of quality is obtained when tested in more than one parameter. In the choice of mates, birds display their quality through several signals such as dancing, colors and vocalizations16. This may be also the reason why more than one neurotransmitter participates in the selection of neuronal connections. Indeed, most synapses depend on more than one neurotransmitter in order to function58.\n\nSeveral observations support the notion of the importance of neurotransmitters in the selection of synapses: glutamate signaling in the auditory system is essential for the normal development of inhibitory circuits, in which some synapses are strengthened and others are silenced57. Glutamate is also important in the maturation of neuronal pathways in the mushroom bodies of Drosophila through non-synaptic mechanisms59. GABA is similarly involved in the development of neuronal circuits through non-synaptic mechanisms60.\n\nIf, as we suggest, released neurotransmitters represent the phenotypic qualities of the releasing cell, the fact that specialized CNS centers release a specific combination of neurotransmitters implies that the neurons in these centers have distinct metabolic activities that relate to the function of the center. For example, in the raphe nuclei, the main source of serotonin in the brain, there is a high extracellular concentration of serotonin, the source of which is a non-synaptic release which is correlated with the activity level of the raphe nuclei61. We suggest that the release of serotonin was adopted, and still functions as, a paracrine signal between cells in the raphe nuclei that facilitates, by a selection process, a local coordination of activity.\n\nNeurons within a specialized population of cells vary in their morphology, their proximity to the sources of metabolites or to incoming stimuli from outside the center, and may vary also with many other parameters62. The specific neurons that are phenotypically more capable to carry out their function are those that react to and process the information received in the center, defining the output of the center. For instance, soma size determines electrophysiological differences between neurons of retinal ganglions, larger neurons having greater excitability63.\n\nIt is reasonable to assume that these phenotypic differences that relate to metabolite capability also determine the level of neurotransmitter released by neurons in the ganglion: less active phenotypes cannot counter the toxic effects of the serotonin released by the more active phenotypes, and consequently lower their metabolism in order to reduce the concentration of serotonin around their outer membrane. Indeed, the release of serotonin in the raphe nuclei is reduced by an increase in its extracellular concentration61, which, we suggest, is a consequence of reduced activity in neurons that reduce their release. If serotonin was not toxic, the more active phenotypes, which produce and release higher concentrations of serotonin, would not reduce the synthesis of serotonin in less active phenotypes, and serotonin release could not serve as a mechanism of selection.\n\nFurthermore we speculate that if the activity of a specific brain center entails the production of a particular waste product, this waste may serve at synapses as an optimal neurotransmitter to ensure that the information provided by the electrical stimulus originates in a specific center.\n\nThe blood-brain barrier of vertebrates separates the extracellular environment of neurons in the CNS from changes caused in peripheral tissues64. It has been suggested that the blood-brain barrier facilitates the maintenance of the highly regulated microenvironment of the synapse by preventing neurotransmitters synthesized in the periphery from reaching synapses in the CNS, creating a “cross-talk” between peripheral and neuronal signaling65 . We suggest, in addition, that if neurotransmitters test and therefore represent the metabolic activity of neurons, then any influx of neurotransmitters from the periphery into the CNS could potentially interfere with that function. In other words, the extracellular concentration of neurotransmitters can only reliably reflect the metabolism of neurons if it is isolated from neurotransmitters produced in the periphery. This may constitute an additional adaptive significance for the mechanisms that prevent toxic neurotransmitters from diffusing through the blood-brain barrier.\n\nAging is accompanied by changes in neurotransmitter concentrations in the brain, and in a number of regions there is a significant decrease in the concentration of glutamate, dopamine and serotonin66–69 . It is possible to interpret the depletion of certain neurotransmitters in old age as an adaptive response to the reduced ability of aging cells to counter the toxicity of these neurotransmitters. Under such conditions it is preferable to reduce the severity of the test rather than to forgo the test altogether. Indeed, dopamine synthesis is regulated by the redox state of the cell, and oxidative stress leads to an inhibition of tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of dopamine70,71. This might explain why restoring the toxicity through an increase in the concentration of certain neurotransmitters, in cells that cannot counter this toxicity, may cause long-term damage, as in the case of l-DOPA treatment for Parkinson’s disease72, while treatment with anti-oxidants has the potential to restore neurotransmitter concentrations to normal levels73.\n\nIt has already been suggested by Le-Corronc et al.74 that the developmental role of neurotransmitters as paracrine signals precedes their role as facilitators of synaptic transmission. Our evolutionary perspective suggests that neurotransmitters that functioned in the periphery as paracrine signals, released directly from the cytoplasm, were initially adopted by the CNS to serve as paracrine signals within specialized CNS centers. The toxicity of the neurotransmitters facilitated the selection of the optimal cells for the particular function of the CNS and coordinated the activity of cells within specific CNS centers. The use of these neurotransmitters at synaptic contacts was later adopted as a signature that identifies the origin of the electrical stimulus arriving at the post synaptic neuron, and prevents other electrical stimuli from interfering with the stimuli from the pre-synaptic neuron.\n\nWe hope that further studies of the function of a CNS center in relation to its particular metabolism involved in processing information may lead to a greater understanding of the relationship between the activity of neurons within the center, and the specific composition of the neurotransmitters they release.\n\n\nAn evolutionary model of the stages that selected toxic chemicals as signals\n\nOur evolutionary perspective suggests that toxic waste released into the extracellular environment by the signaling cell, a release that is inherently correlated to the activity of the signaling cell, forces neighboring cells to react to counter the toxicity of the release. Their reaction may provide them with information that can contribute to the coordination of their activity with neighboring cells. Here we explain the model in the context of various examples that were instructive in its development.\n\nDifferent metabolic activities result in the production of particular waste products. For example, oxidative phosphorylation in mitochondria leads inevitably to the production of reactive oxygen species75. Another example is the release of ACh, which is correlated to calcium influxes76: as motor activities require the influx of calcium ion into the cytoplasm77, and as ACh is also a positive ion, its release is an inevitable result of the influx of calcium ions76. While other positive ions may be released as a result of the influx of calcium, ACh is quickly hydrolyzed outside the cell78, as opposed to inorganic ions, and therefore reliably reflects in more detail than other ions the current activity of the releasing cell.\n\nIt is also reasonable to assume that the level of the waste released is correlated to the level of the activity of the releasing cell, such as the correlation between carbon dioxide production and the level of respiration79.\n\nAmong the waste products released, some are more toxic and potentially harmful to nearby cells, since waste released within a multicellular organism encounters the outer cell membrane of nearby cells in addition to its potential harm to the signaling cell.\n\nCells exposed to a toxic chemical must counter the toxicity via (1) producing and releasing anti-oxidants, such as the release of ascorbate to reduce dopamine-mediated oxidative damage37, (2) degrading the chemical enzymatically, such as acetylcholinesterase78, or (3) transporting the chemical into the cytoplasm where it can be converted into less harmful chemicals or transported into and stored inside vesicles, as in the case of glutamate and dopamine5,24.\n\nThe uptake of glutamate or the release of antioxidants which counters the toxicity of dopamine is correlated to their respective concentrations outside the cell. The response to a toxic chemical must be related to its concentration if it is to counter its toxicity. In addition, the toxicity also harms the membrane of the releasing cell, limiting its metabolic activity in order to prevent the cell from increasing the level of release beyond its ability to cope with the toxicity, as evidenced by the inhibition of serotonin secretion and synthesis by extracellular serotonin61.\n\nConsequently, the activity of a cell to counter the toxicity of chemicals in its extracellular environment can provide it with information on its potential to be active as compared with that of the secreting cells. Such information can serve as a cue to facilitate the coordination of activities with those of the releasing cell, for instance, in the course of the development of osteoblasts that is mediated by glutamate80, to either differentiate, undergo mitosis or apoptosis. Coordination between neighboring cells is necessary within multicellular organisms, and we suggest that the information provided by the reaction to released toxic waste can facilitate this coordination: for instance, in airway epithelium, which coordinates cilia beating via ACh81, or in developing tissues such as developing osteoblasts, which coordinate development via glutamate signaling82.\n\nBefore the organism benefited from the reaction of neighboring cells to the release of the toxic chemical, mutations that resulted in increased synthesis of the released toxic chemical would have been detrimental. However, once neighboring cells became attentive to changes in the level of the released chemical, the organism could benefit from enzymes that increase the production of the toxic chemical in the releasing cell, which can provide more detailed and accurate information about a change in its metabolism, and facilitate the synchronization of activities between neighboring cells.\n\nThis extra investment in increasing the production of a toxic chemical (the handicap), changes the released chemical from a cue into a signal, and provides the basis for a paracrine signaling system16,83. We follow Maynard Smith and Harper18 in defining a signal as a trait that benefits the signaler only if the receiver reacts to it in a way that benefits the signaler.\n\nIt is interesting to note that the CNS uses ACh to stimulate peripheral cells, which is the same signal that is used in the periphery in paracrine signaling, rather than evolving a novel neurotransmitter, a process that would require the coevolution of receptors and complementary transduction systems to process the information. It is possible that the release of ACh from myocytes84, which we suggest is an inevitable result of calcium influx, can serve as a paracrine signal and as a retrograde signal that provides reliable information regarding myocyte contraction to extrasynaptic ACh receptors on the motor neuron83. It is possible that other neurotransmitters also serve as retrograde signals. For example, glutamate serves as a retrograde signal between cerebellar Purkinje neurons85.", "appendix": "Author contributions\n\n\n\nAll three authors took part in the conception and development of the ideas, and the composition and editing of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe would like to thank David Gurwitz for his insightful comments on the content and presentation of the manuscript, Daniel Offen, Ari Barzilai and Vidyanand Nanjundiah for their comments during the preparation of the manuscript, and Naomi Paz for stylistic improvements.\n\n\nReferences\n\nWrona MZ, Yang Z, Zhang F, et al.: Potential new insights into the molecular mechanisms of methamphetamine-induced neurodegeneration. NIDA Res Monogr. 1997; 173: 146–174. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "6204", "date": "22 Sep 2014", "name": "Ulrich Technau", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper proposes a somewhat provocative but also inspiring hypothesis which claims that neurotransmitters evolved from a similar principle as the sexual signals in birds: it tests the activity and the status of the signaling cells by secreting a toxic substance. They provide an overview of the toxicity of neurotransmitters when concentrations are slightly unbalanced. They also propose that the blood-brain barrier evolved as part of the distinct signaling of neurons in the CNS, without disturbance by signals produced in the periphery. The authors provide interesting thoughts as to why and how the transmitter system could have evolved from the release of a toxic waste. This is all fine, but what I miss is the evolutionary perspective promised in the title, which not only is based on a \"Gedankenexperiment\" but on available evidence. All animals except sponges and placozoans have neurons. Current evidence suggest that neurons from cnidarians and bilaterians have a common origin, which is also reflected by the use of the same transmitters (although the Hydra genome shows that several crucial genes of Ach production are missing). The recent analysis of the ctenophore genome led, however, to the conclusion that neurons evolved independently in ctenophores and bilaterians. Sponges, on the other hand, have many synaptic genes present in the genome, yet lack neurons. In summary, interesting hypothesis, but I miss a discussion of all these available genomic data in the context of the hypothesis.", "responses": [] }, { "id": "6188", "date": "30 Sep 2014", "name": "Rony Paz", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting speculative paper, suggesting a novel explanation for a long-standing question: why are neurotrasmitters toxic? It applies a similar logic and rationale as in the original handicap-principle (and the extended signal selection) to neurotransmitters and their use as signalling system between neurons. As such, it suggests a nice explanation and the authors supply several examples that this approach can help explain. Yet it also suffers from the lack of more conclusive evidence, as many other evolutionary-driven explanations. I would ask the authors to suggest direct predictions that can be tested in an experimental setup, and supply few examples of putative results that might argue against their idea. If such predictions are provided, the paper will be strengthened and would constitute an important idea.", "responses": [ { "c_id": "1090", "date": "21 Nov 2014", "name": "Keith Harris", "role": "Author Response", "response": "We have added in the new revision a suggestion of how our hypothesis might be tested fairly simply, and what results would argue against our idea." } ] } ]	1	https://f1000research.com/articles/3-179
https://f1000research.com/articles/3-293/v1	02 Dec 14	{ "type": "Clinical Practice Article", "title": "Slow progression of exudative age related macular degeneration associated with hypertrophy of the retinal pigment epithelium", "authors": [ "Jeffrey Stern", "David Eveleth", "Jennifer Masula", "Sally Temple", "David Eveleth", "Jennifer Masula", "Sally Temple" ], "abstract": "Rationale: Choroidal neovascular (CNV) lesions in younger patients are often accompanied by the appearance of a surrounding ring of pigment that is associated with disease regression or slowed disease progression. In older patients with age-related macular degeneration (AMD), however, hypertrophy of the retinal pigment epithelium (RPE) is known to occur but has not previously been reported to be associated with CNV regression. This report describes the clinical course of a case series of AMD patients with pigment hypertrophy adjacent to CNV associated with stabilization of the CNV lesion.Methods: A retrospective analysis of exudative AMD patients seen by a single retina specialist over a 7-year period.Results: Retrospective analysis of 955 exudative AMD patients revealed pigment hypertrophy associated with CNV in 33 patients. A ring of pigment surrounded CNV in 6 of these. Three representative patients are presented to illustrate the decrease in macular edema, reduced fluorescein leakage and slowed CNV progression that was associated with a pigment ring around CNV in AMD. Pigment hypertrophy was associated with blocked fluorescein leakage and exudative AMD patients with a complete pigment ring maintained stable visual acuity, macular edema, fluorescein leakage and CNV lesion size without treatment for intervals of up to 21 months. Conclusion: We report slowed disease progression in AMD patients who develop pigment around CNV. The slow rate of disease progression in the AMD patient subgroup having a pigment ring is a factor to consider in determining the treatment interval for exudative AMD patients.", "keywords": [ "macular degeneration", "AMD", "choroidal neovascularization", "CNV", "retinal pigment epithelium", "RPE", "AMD treatment interval", "RPE wound healing" ], "content": "Introduction\n\nA rapid loss of vision in exudative age-related macular degeneration (AMD) occurs when choroidal neovascular membranes (CNV) grow into the overlying retinal pigment epithelium (RPE) and neurosensory retina. The natural course of CNV is generally continued growth until central vision is lost, with rare spontaneous resolution in exudative AMD1. In contrast, young patients with CNV secondary to myopia2, histoplasmosis3, rubella4 or other causes5,6 often undergo stabilization that is accompanied by pigment hypertrophy developing around the CNV lesion. Although pigment hypertrophy is well known to occur in AMD patients1,7, there have been no reports on pigment hypertrophy associated with CNV regression in AMD. Here we report 3 AMD patients who developed a ring of hyperpigmentation around CNV during treatment of the CNV lesion that was accompanied by CNV regression even after the treatment was withdrawn for periods of up to 21 months.\n\n\nMaterials and methods\n\nPigment hypertrophy was noted during fundus photography and fluorescein angiography (FA) for exudative AMD patients seen by a single retina specialist over a 7 year period. From a total of 966 exudative AMD patients, 33 developed a ring of pigment around the CNV lesion. Written informed consent to show images for research purposes was obtained from these patients. A prominent ring of pigment in the absence of significant hemorrhage or fibrous proliferation was observed in 6 of the 33 patients, and 3 had an uninterrupted series of fundus, fluorescein angiography (FA) and optical computed tomography (OCT) images suitable for presentation. Fundus photographs and FA images were obtained with a Zeiss FF450 or Topcon 50X fundus camera and OCT images were obtained with Zeiss, Optos or Heidleberg devices. Treatments were standard clinical practice in the year that care was provided, which included thermal laser, visudyne photodynamic therapy (PDT) and anti-vascular endothelial growth factor (anti-VEGF) intravitreal injections of bevacizumab (Avastin), pegaptanib (Macugen) or ranibizumab (Lucentis). Treatment intervals were varied as described for each case.\n\n\nDescription of cases\n\nThe first case is a 58 year old woman who presented with metamorphopsia and worsening visual acuity to 20/30. The fundus photograph in Figure 1A, mid-phase FA in Figure 1A’, magnified FA in Figure 1A”; and OCT image in Figure 1A* were taken prior to treatment. These indicate perifoveal CNV with fluorescein leakage and serous detachment. The CNV was initially treated with focal thermal laser and the patient remained stable for 6 months, after which CNV recurred toward the fovea at the supero-temporal edge of the laser scar. Combined therapy with 4 bevacizumab injections (1.25mg/0.05ml) and 2 full dose visudyne PDT treatments were delivered over a 6-month period during which a pigment ring formed to partially surround the CNV lesion (Figures 1B-B). The patient then remained stable for 9 months without treatment. Following this period, serous fluid accumulated under the fovea with CNV recurring toward the original thermal laser scar in a direction away from the pigment ring (Figures 1C-C). This second CNV recurrence was treated with bevacizumab (1.25mg/0.05ml) or ranibizumab (0.5mg/0.05ml) every 4–6 weeks for 9 months during which time fluorescein leakage remained blocked in the direction of the pigment ring and active in the direction of the original thermal laser scar. The horizontal OCT images through the CNV lesion (Figures 1C-D) indicate that serous fluid is replaced by RPE layer thickening. After 42 months of anti-VEGF therapy, fluorescein leakage remained contained in the direction toward the pigment ring with active leakage in the direction away from pigment.\n\nFundus photograph (A–D), fluorescein angiogram (FA) mid-phase (A’–C’), magnified FA (A”–D”) and OCT (A–D) images from a 58 year old female patient presenting with worsening visual acuity to 20/30 and metamorphopsia. Figures 1A-A) Images taken prior to treatment show a peri-foveal fluorescein leakage with serous detachment due to CNV. The patient was treated with thermal laser to ablate the CNV lesion. Figures 1B-B) After 6 months, CNV recurred in the supero-temporal aspect of the laser scar. The recurrence was treated with combined therapy of 2 PDT and 4 bevacizumab injections applied over a 6 month period. Figure 1C-C) After remaining stable for 9 months without treatment, the CNV again recurred in the direction of the thermal laser scar but not in the direction of the partial pigment ring. This recurrence was treated with serial anti-VEGF antibody injections and then remained stable after 42 months with anti-VEGF therapy as shown in Figure 1D-D.\n\nThe second patient is a 71 year old woman who presented with metamorphopsia and decreased acuity to 20/50. Initial fundus photography and FA indicated a perifoveal classic CNV (Figures 2A-A”). The CNV was treated with 4 full standard visudyne PDT sessions over a 13 month period. During this time, a ring of pigment formed to completely surround the CNV (Figures 2B-B”). The patient then remained stable for 20 months without treatment after which symptoms and fluorescein leakage recurred (Figures 2C-C”). Of note, the minimal leakage recurred where the pigment ring remained intact compared to more extensive fluorescein leakage where CNV broke through the infero-nasal aspect of the pigment ring. The patient then received 31 months of anti-VEGF therapy which resulted in the reformation of a complete pigment ring and deceased leakage (Figures 2D-D). The CNV lesion remained stable with minimal leakage and visual acuity stable at 20/60 for 21 months without treatment (Figures 2E-E).\n\n(Figures 1A-A’) shows a 71 year old female patient who presented with decreasing vision over 1 week with fundus photography and angiography indicating perifoveal CNV and a small amount of hemorrhage. Figures 2B-B”) After 5 visudyne PDT treatments over a 13 month period, a complete ring of pigment formed to surround the CNV and treatment was withheld. Note the reduced fluorescein leakage. Figures 2C-C’) The treatment interval was extended to 20 months after which symptoms and a small infero-temporal area of fluorescein leakage recurred. Anti-VEGF treatments with bevacizumab and pegaptanib were initiated and after 7 treatments over a 31 month period, a pigment ring re-formed around the CNV with elimination of the infero-temporal leakage as shown in Figures 2D-D. After this, the treatment interval was again extended to 21 months without treatment during which the lesion remained stable as shown in Figures 2E-E.\n\nThe third patient is a 48 year old woman who presented with metamorphopsia and vision loss to 20/40. The initial findings indicated CNV adjacent to the fovea (Figures 3A-A”) for which anti-VEGF therapy with bevacizumab was initiated. After 5 injections over a 38 week period, leakage diminished and a dense pigment ring formed around the lesion (Figures 3B-3B’). Treatment was withheld and the patient remained stable for 10 months, but then complained of increasing metamorphopsia and was found to have recurrent CNV. The treatment was re-initiated for a 31 month period after which the pigment ring reformed and the treatment interval was again extended. The patient then remained stable with complete pigment capping, acuity of 20/20, and minimal FA leakage for 10 months without treatment (Figures 3C-C) after which she was lost to follow-up.\n\n(Figures 3A-A”) show the initial fundus photographs and FA taken from a 48 year old woman presenting with metamorphopsia decreased visual acuity and a small, subfoveal CNV. After 5 anti-VEGF treatments with bevacizumab over a 9 month period, a pigment ring formed containing the leakage and treatment was withheld for 10 months until a small recurrence occurred toward the fovea as shown in Figures 3B-B. Anti-VEGF treatments with bevacizumab were re-initiated and after 7 treatments over a 31 month period, a pigment ring re-formed around the CNV with elimination of the infero-temporal leakage. This was followed by a 10 month interval without treatment, during which the CNV again remained quiescent with a surrounding pigment ring as shown in Figures 3C-C.\n\n\nDiscussion\n\nIn this case series, 3 AMD patients developed a ring of pigment around CNV which was accompanied by decreased fluorescein leakage and slowed CNV growth in the direction of the pigment. A complete pigment ring was associated with much less rapid disease progression than expected1, and patients with a complete ring were stable without treatment for extended periods. The presence of a pigment ring is known to be associated with CNV regression in younger patients2–4. Our case series suggests that the presence of pigment hypertrophy surrounding CNV can also be associated with slowed growth or regression of CNV in patients with AMD. We suggest that the presence of a pigment ring be considered in determining the treatment interval offered to AMD patients.\n\nWound repair of central nervous system tissues such as the RPE and neural retina is generally limited, yet evidence for RPE proliferation and wound repair has been described in young patients with CNV2–6, after RPE rips8,9, in animal models of laser-induced CNV10, after RPE debridement11, and to repopulate areas of RPE loss in vitro12. It is possible that a proliferative RPE response to CNV has only recently become evident in AMD patients due to a prior lack of treatment to slow CNV growth that otherwise overwhelms the RPE response. The advent of new therapies to slow CNV growth may have altered the balance between CNV and RPE to unmask RPE wound healing. In both younger and older patients, increased pigmentation and thickening of the RPE layer is consistent with the hypothesis that CNV elicits a proliferative response in the RPE layer that strengthens the barrier against further CNV invasion. This RPE layer self-repair may be mediated by activation of a subpopulation of RPE stem cells that has been recently identified12.\n\n\nConsent\n\nWritten informed consent to publish clinical images has been obtained from each patient.", "appendix": "Author contributions\n\n\n\nThe data were collected by JS and JM, and analyzed by JS, JM, DE and ST. The manuscript was written by JS, DE and ST. All authors agreed to the final content.\n\n\nCompeting interests\n\n\n\nDrs. Stern, Eveleth and Temple are shareholders in Athghin Biotechnology, Inc.\n\n\nGrant information\n\nThe authors are grateful for funding from the NIH-NEI Audacious Goals Prize (JS).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe are grateful to Hao Wang, M.D. for reading the manuscript. This study utilized support from the NEI Audacious Goals Prize (JS).\n\n\nReferences\n\nGass JDM: Stereoscopic Atlas of Macular Diseases: Diagnosis and Treatment. 4th Edition. Mosby, St Louis, Mo. 1997; 49–286. Reference Source\n\nAvila MP, Weiter JJ, Jalkh AE, et al.: Natural history of choroidal neovascularization in degenerative myopia. Ophthalmology. 1984; 91(12): 1573–1581. PubMed Abstract \| Publisher Full Text\n\nKleiner RC, Ratner CM, Enger C, et al.: Subfoveal neovascularization in the ocular histoplasmosis syndrome: A natural history study. Retina. 1988; 8(4): 225–9. PubMed Abstract \| Publisher Full Text\n\nVeloso CE, Costa RA, Oréfice JL, et al.: Spontaneous involution of choroidal neovascularization secondary to rubella retinopathy. Eye (Lond). 2007; 21(11): 1429–30. PubMed Abstract \| Publisher Full Text\n\nCampochiaro PA, Morgan KM, Conway BP, et al.: Spontaneous involution of subfoveal neovascularization. Am J Ophthalmol. 1990; 109(6): 668–675. PubMed Abstract \| Publisher Full Text\n\nHo AC, Yannuzzi LA, Pisicano K, et al.: The natural history of idiopathic subfoveal choroidal neovascularization. Ophthalmology. 1995; 102(5): 782–9. PubMed Abstract \| Publisher Full Text\n\nGrossniklaus HE, Gass JD: Clinicopathologic correlations of surgically excised type 1 and type 2 submacular choroidal neovascular membranes. Am J Ophthalmol. 1998; 126(1): 59–69. PubMed Abstract \| Publisher Full Text\n\nChuang EL, Bird AC: Repair after tears of the retinal pigment epithelium. Eye (Lond). 1988; 2(Pt 1): 106–113. PubMed Abstract \| Publisher Full Text\n\nPeiretti E, Iranmanesh R, Lee JJ, et al.: Repopulation of the retinal pigment epithelium after pigment epithelial rip. Retina. 2006; 26(9): 1097–1099. PubMed Abstract \| Publisher Full Text\n\nMiller H, Miller B, Ryan SJ: The role of retinal pigment epithelium in the involution of subretinal neovascularization. Invest Ophthalmol Vis Sci. 1986; 27(11): 1644–1652. PubMed Abstract\n\nLopez PF, Yan Q, Kohen L, et al.: Retinal pigment epithelial wound healing in vivo. Arch Ophthalmol. 1995; 113(11): 1437–1446. PubMed Abstract \| Publisher Full Text\n\nWang H, Ninomiya Y, Sugino IK, et al.: Retinal pigment epithelium wound healing in human Bruch’s membrane explants. Invest Ophthalmol Vis Sci. 2003; 44(5): 2199–2210. PubMed Abstract \| Publisher Full Text\n\nSalero E, Blenkinsop TA, Corneo B, et al.: Adult human RPE can be activated into a multipotent stem cell that produces mesenchymal derivatives. Cell Stem Cell. 2012; 10(1): 88–95. PubMed Abstract \| Publisher Full Text" }	[ { "id": "7253", "date": "26 Jan 2015", "name": "Thomas Gardner", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have extended prior clinical observations of RPE rings associated with resolution of sub retinal neovascularization in young patients and now find 3 unusual cases of similar findings in patients with AMD. These findings represent astute clinical observations and the combination with possible cellular pathophysiology and potential implications for treating AMD make this a worthy manuscript for consideration. The title and abstract are appropriate, the writing is clear and succinct, and the observations will be of use in the field. In my own observations I concur that the presence of RPE rings is a likely correlate of CNV regression.", "responses": [] }, { "id": "7254", "date": "03 Feb 2015", "name": "Igor Nasonkin", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article by Dr. Stern and colleagues is focused on summarizing cases of wet AMD with CMV lesions where Retinal Pigment Epithelium (RPE) undergoes self-repair associated with hypertrophy, slower disease progression and a ring of pigment forming around the CNV, caused by RPE proliferation.This paper should be of interest to both clinicians and basic researchers working in regenerative ophthalmology. Age Related Macular Degeneration is the leading cause of vision loss in the world, and wet AMD pathology is very destructive to the fine layer of neural retina and RPE. While anti-VEGF antibody (bevacizumab) intravitreal injections is a powereful treatment to slow down vision loss caused by wet AMD, there is no regeneration of RPE at the site of initial CMV lesion and RPE tissue in this part of retina is considered to be lost forever. Cell transplantation (such as RPE derived form adult or embryonic stem cells) is one innovative way to approach RPE repair and treating vision loss in wet AMD. However, cell transplantation is a very invasive procedure per se and may precipitate further destruction of the fine and very sensitive layer of neural tissue such as neural retina/RPE. Finding innovative ways to induce the regeneration of RPE in patients with AMD will likely be a significant leap forward in treating this devastating disease.Dr. Stern's 7 year old summary of observing patients with wet AMD and CNV identified a very interesting group of about 3.5 % of patients (33 out of 955 total), where spontaneous RPE proliferation took place, associated with a distinct ring of pigment around the CMV, decreased macular edema and stabilized vision. This indicates that some small percentage of patients are able to partially regenerate RPE at the site of lesion after initial RPE damage took place, and provides a unique opportunity to identify the factors (secreted by CMV lesion, as well as genetic factors) enabling this selected cohort of patients to initiate RPE regeneration (hypertrophy).Though the response leading to the formation of so called pigment ring, does not lead to substantial regeneration of RPE, this nevertheless leads to stabilization of vision and does slow down the disease progression. The report gives a unique tool to regenerative ophthalmology, as well as basic researchers focused on RPE biology, pathology and regeneration to identify such factors and pathways, which are activated in RPE in this cohort of patients, and enable RPE to reenter cell division and replenish and repair the RPE layer damaged by AMD. The next logical step seems to be finding the small molecules, which may mimic CMV signaling and initiate RPE proliferation in situ. Dr. Stern's report provides unique tools to scientists working on AMD and retinal regeneration to find the innovative approaches to restore retina by stimulating the endogenous regeneration in adult RPE or/and in subpopulation of cells within RPE layer, which possesses stem cell signature and capable to better respond to proliferative signals from CMV lesion.Collectively, this report is of substantial value and significance and should be indexed to enable researchers to find the mechanisms, controlling the endogenous regeneration of RPE.", "responses": [] } ]	1	https://f1000research.com/articles/3-293
https://f1000research.com/articles/3-214/v1	09 Sep 14	{ "type": "Software Tool Article", "title": "ABS–Scan: In silico alanine scanning mutagenesis for binding site residues in protein–ligand complex", "authors": [ "Praveen Anand", "Deepesh Nagarajan", "Sumanta Mukherjee", "Nagasuma Chandra", "Praveen Anand", "Deepesh Nagarajan", "Sumanta Mukherjee" ], "abstract": "Most physiological processes in living systems are fundamentally regulated by protein–ligand interactions. Understanding the process of ligand recognition by proteins is a vital activity in molecular biology and biochemistry. It is well known that the residues present at the binding site of the protein form pockets that provide a conducive environment for recognition of specific ligands. In many cases, the boundaries of these sites are not well defined. Here, we provide a web-server to systematically evaluate important residues in the binding site of the protein that contribute towards the ligand recognition through in silico alanine-scanning mutagenesis experiments. Each of the residues present at the binding site is computationally mutated to alanine. The ligand interaction energy is computed for each mutant and the corresponding ΔΔG values are computed by comparing it to the wild type protein, thus evaluating individual residue contributions towards ligand interaction. The server will thus provide clues to researchers about residues to obtain loss-of-function mutations and to understand drug resistant mutations. This web-tool can be freely accessed through the following address: http://proline.biochem.iisc.ernet.in/abscan/.", "keywords": [ "Currently (as of April 3", "2014)1 there exist more than 72000 (as of April 3", "2014) experimentally determined protein structures complexed with small molecule ligands", "providing an extensive data resource on protein binding sites. These binding sites vary in size ranging from six to thirty residues depending upon the size and the nature of the ligand. In most cases", "the contribution of the individual amino acids towards the binding of a given ligand is not well understood. A well-established method of demonstrating the importance of a residue at the site is to create point mutants through site-directed mutagenesis2. Efforts towards characterization of entire functional site include tools such as alanine scanning mutagenesis (ASM)3", "where each residue is mutated to an alanine and its effect on the function is evaluated. ASM is indeed a well-used technique in experimental biology and has been successfully applied to the problems of protein folding and stability4", "protein-protein5", "6", "and protein-ligand7 interactions. The experimental success of this technique has resulted in further developments", "including high-throughput and low-cost variants8", "greatly expanding its reach. Yet", "given the time", "cost and effort required for carrying out experimental biochemistry", "a large majority of proteins are yet to be studied through this method." ], "content": "Introduction\n\nCurrently (as of April 3, 2014)1 there exist more than 72000 (as of April 3, 2014) experimentally determined protein structures complexed with small molecule ligands, providing an extensive data resource on protein binding sites. These binding sites vary in size ranging from six to thirty residues depending upon the size and the nature of the ligand. In most cases, the contribution of the individual amino acids towards the binding of a given ligand is not well understood. A well-established method of demonstrating the importance of a residue at the site is to create point mutants through site-directed mutagenesis2. Efforts towards characterization of entire functional site include tools such as alanine scanning mutagenesis (ASM)3, where each residue is mutated to an alanine and its effect on the function is evaluated. ASM is indeed a well-used technique in experimental biology and has been successfully applied to the problems of protein folding and stability4, protein-protein5,6, and protein-ligand7 interactions. The experimental success of this technique has resulted in further developments, including high-throughput and low-cost variants8, greatly expanding its reach. Yet, given the time, cost and effort required for carrying out experimental biochemistry, a large majority of proteins are yet to be studied through this method.\n\nDue to availability of a variety of structural bioinformatics tools, it is now feasible to carry out alanine scanning mutagenesis computationally9. Spurred by the successes and widespread adoption of the ASM technique, various computational resources now exist for in-silico alanine scanning. Prominent examples include Modeller10 and the Rosetta software suite11. However, most packages are command-line oriented and are out of reach for researchers. Alanine scanning webservers with intuitive user interfaces such as Robetta webserver12, the Rosetta Design web-server13, ROSIE14, FOLDX15, BeATMuSiC16, exist for the problems of protein folding, protein stability and protein-protein interactions. Although, there are workflows to evaluate ligand-binding energetics which require significant computational time and setup through free-energy calculations involving Molecular Mechanics/Generalized Born Surface Area method (MM-GBSA)17,18, there is however, no intuitive web-tool available for analyzing alanine-scanning mutations of small-molecule binding site residues in real time. A common requirement for an experimental biochemist is to identify which amino acids to mutate in the protein to generate loss-of-function mutants. A web-tool to cater to that specific need will therefore be highly useful. The analysis will also provide deep insights into critical residues for interaction, residue pairs or sets that when mutated will abolish ligand binding and provide analytical insights for lead refinement in the process of drug discovery, as well understand drug resistance due to mutations.\n\nWe present a computational workflow and webserver, Alanine Binding Site-Scan (ABS-Scan), for automated alanine-scanning mutagenesis of protein-ligand interface residues. The workflow combines the libraries of widely used software packages including Modeller10 for site-specific alanine mutagenesis and Autodock19 for energetic evaluation of protein-ligand complexes.\n\n\nWorkflow\n\nThis workflow allows a user to submit a protein-ligand complex of their interest (Figure 1). The user is provided with an option of selecting a distance cut-off to define the binding site around a specific ligand for which, in-silico alanine scanning mutagenesis is carried out. Once the input parameters are obtained, the Modeller library is used to perform site-specific mutagenesis on all selected residues, coupled with steps of energy minimization. Each mutated structure, will then be scored by using Autodock 4.1 force field, to calculate the energetics of a protein-ligand complex. The essentiality of a residue can be determined by difference in interaction score of mutant and wild-type protein (∆∆G value). These results are graphically presented to the user, along with a ranked list of residues in the given site that could be experimentally explored for site-directed mutagenesis. A Jmol applet displays protein-ligand interactions with residues colored according to the computed extents of contribution towards interaction, while a table simultaneously displays inter-molecular energy scores. We also provide a help-section explaining the results along with selected examples.\n\nFlowchart depicting various steps involved in ABS-Scan.\n\n\nValidation\n\nMainly two types of validation were carried out, first to find a correlation with experimentally determined binding affinities followed by sensitivity evaluation of the predicted ABScan ∆∆G scores. The first exercise involved systematically mining the available experimental information related to alanine-scanning mutagenesis of binding site residues. A methodical search was carried out to mine all the experimental results available in literature on alanine-scanning mutagenesis of residues at the binding site. Advanced search option in PDB was used for this purpose. All the PUBMED extracts were scanned for the term - “alanine scanning”. The results obtained were further filtered to contain only X-Ray experimental data, and abscence of any DNA, RNA or DNA/RNA hybrid in the PDB entity. The results were further restricted to only those entries that had ligands bound to them, and we expected that this would reduce the hits that contain alanine-scanning mutations for evaluating protein-protein interfaces. The above search criteria mentioned yielded 126 structure hits with 56 citations. The list of entries obtained, was further pruned to remove biologically irrelevant ligands, metal ions and modified residues. The list of 79 entries that we finally obtained can be accessed at http://proline.biochem.iisc.ernet.in/abscan/validation. Each of the above experiments involving alanine-scanning mutagenesis reports different mutant evaluation scores. The measures reported to test the fitness of the mutants include various attributes such as Kd, Ka, kcat/KM (for enzymes), specific substrate/product assays etc. These measures cannot be normalized to derive values having uniform units for direct comparison. We picked a few of the examples to see the correlation between experimentally reported mutant evaluation scores and the predicted ∆∆G values. One such example has been described here.\n\nA study on testosterone binding site of rat 3-alpha-hydroxysteroid dehydrogenase (PDBID: 1AFS) by Heredia et al.20 reports that binding site residue in direct contact with the ligand influences the rate determining step of the enzymatic reaction. Alanine scanning mutagenesis was performed on binding site residues of the hydroxysteroid dehydrogenase protein that could interact with the steroid ligand and Kd was experimentally determined for each mutant to prove this. The ABS-Scan analysis performed on this complex with both testosterone and progesterone also confirms this. A good correlation was observed between the reported Kd value and the corresponding ∆∆G score predicted by ABS-Scan (Figure 2). The details of the experimental values, predicted score and the web-server output can be visualized along with other examples at http://proline.biochem.iisc.ernet.in/abscan/validation.\n\nGood agreement is observed between experimental Kd values and predicted ∆∆G values determined for (A) testosterone & (B) progesterone binding site alanine scanning mutagenesis performed on rat 3-alpha-hydroxysteroid dehydrogenase.\n\nIn order to determine the sensitivity of ABS-Scan, we compared predictions of essential residues through ABS-Scan in native complexes with corresponding decoy complexes. The complete dataset was obtained from the Community Structure-Activity Resource (CSAR - www.csardock.org/). Decoys in this dataset contain artificial docked complexes of protein with ligands having similar chemical properties to native ligands, but not known to interact with the protein. ABS-Scan is seen to effectively discriminate between the decoy and the native complexes (p-value ~0.004 calculated with Student’s t-test) in ~67% of the cases (∆∆G ≥0.5). This clearly indicates that residues important for ligand interaction can be identified through our approach (Figure 3). The details of validation protocol and results are accessible from the web-resource at http://proline.biochem.iisc.ernet.in/abscan/validation.\n\nBoxplot showing the difference in the % of the residues in the binding site of cognate and decoy dataset having a predicted ∆∆G score ≥0.5.\n\n\nImplementation\n\nThe webserver was implemented using hypertext preprocessor (PHP). Autodock, Modeller and Pymol libraries have been used for modeling the mutation and evaluating the energetics. Integration of these back-end libraries for presentation as a functional and intuitive user interface is accomplished using Shell, Python, Java, HTML and PHP scripts. The web-server is platform independent and will run on any machine having internet access with browser installed. For the advanced users, a command-line interface in the form of a single python script can be accessed from github repository (https://github.com/praveeniisc/ABS-Scan). The script has been tested on Intel 2.83 GHz quad-core system running 32 bit linux OS(Ubuntu 12.04) with Modeller10, MGL AutodockTools19 & Pymol (http://pymol.org) installed. For the web-server d3.js library has been used for displaying the plots. Jmol Applet has been used to visualize the protein-ligand interaction.\n\nThe input required for the server is the structure of protein-ligand complex in PDB format. Users can either provide the four-letter PDBID or upload the PDB structure file of the complex. An option is provided to define the cut-off distance and select the ligand to obtain binding site residues which would be mutated to alanine for evaluating the interaction energetics. A default distance cut-off of 4.5 Å is set to select all the residues within this distance from any atom of the ligand. In some the cases, metal ions21 and water molecules are observed to play a crucial role in stabilizing the interactions22. Major problem involved in incorporating the ligand metal ion in ABS-Scan worflow is fixing the charge parameter as metal atoms can have different ionic states (Ex. Fe2+, Fe3+ etc.) which is important for evaluating energetics. Enumerating all important structural water molecules involved in the ligand interaction is also highly dependent on the resolution of the crystal structure. Hence, an advanced option is provided to the user for uploading the PDBQT format of the ligand, to account for cases where the ligand contains unusual atom types, metal ions or uses bridge-water molecules for interaction.\n\nAll the results produced by ABScan can be visualized interactively on the web-server. Jmol Applet is used to visualize the contribution of residues towards ligand interaction (Figure 4).\n\nSnapshot explaining the Jmol applet output on the ABScan server. The individual residues are colored in red to blue gradient depending upon the contribution towards the ligand interaction as predicted by ABScan ∆∆G score. Options to visualize the different kinds of interaction - polar, hbonds etc. is also provided.\n\nd3.js library has been utilized to plot the predicted ∆∆G values and subcomponents of the energetic scores reported by Autodock4 (Figure 5). An option is provided to download publication quality images in SVG/PDF/PNG formats. Twitter bootstrap java library is used for framework development on the webserver. An option is also provided to download the raw files containing individual mutants in PDB format, ∆∆G scores in the raw CSV format along with autodock energy scores.\n\n(A) ∆∆G values reported for each of the alanine mutation performed for the residues present at the binding site. The residues are ordered according to their contribution/∆∆G values. (B) The different energy component of autodock interaction score plotted for each of the alanine mutant produced at the binding site.\n\n\nConclusions\n\nABS-Scan webserver can provide valuable insights on molecular recognition involving protein-ligand interactions. Experimentally determined protein-ligand structures can be studied to understand individual residue contributions towards ligand binding. Modeled complexes can also be submitted to infer the feasibility of the interaction. We believe that ABS-Scan would add one more dimension to the analysis of binding sites in proteins, comparison of various ligand interactions and be of importance to researchers performing ASM studies.\n\n\nSoftware availability\n\nhttp://proline.biochem.iisc.ernet.in/abscan/\n\nhttps://github.com/praveeniisc/ABS-Scan\n\nhttps://github.com/F1000Research/ABS-Scan/releases/tag/V1.0\n\nhttp://dx.doi.org/10.5281/zenodo.1142324\n\nABS-Scan is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.", "appendix": "Author contributions\n\n\n\nConceived and designed the experiments: NSC. Performed the experiments: PA,DN,SM. Analyzed the data: PA,DN,SM,NSC. Wrote the paper: PA,DN,NSC. Website design and implementation: PA.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe authors(s) declare that no special grants were sanctioned for this project. PA was supported by Bristol-Myers Squibb fellowship while carrying out this work.\n\n\nAcknowledgements\n\nWe acknowledge all the members of the NSC lab for useful suggestions during the development of the web-server and visualization of the results.\n\n\nReferences\n\nRose PW, Bi C, Bluhm WF, et al.: The RCSB Protein Data Bank: new resources for research and education. Nucleic Acids Res. 2013; 41(Database issue): D475–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMorrison KL, Weiss GA: Combinatorial alanine-scanning. Curr Opin Chem Biol. 2001; 5(3): 302–7. PubMed Abstract \| Publisher Full Text\n\nWeiss GA, Watanabe CK, Zhong A, et al.: Rapid mapping of protein functional epitopes by combinatorial alanine scanning. Proc Natl Acad Sci U S A. 2000; 97(16): 8950–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilliams AD, Shivaprasad S, Wetzel R: Alanine scanning mutagenesis of Abeta(1-40) amyloid fibril stability. J Mol Biol. 2006; 357(4): 1283–94. 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PubMed Abstract \| Publisher Full Text\n\nGreenidge PA, Kramer C, Mozziconacci JC, et al.: MM/GBSA binding energy prediction on the PDBbind data set: successes, failures, and directions for further improvement. J Chem Inf Model. 2013; 53(1): 201–9. PubMed Abstract \| Publisher Full Text\n\nTrott O, Olson AJ: AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem. 2010; 31(2): 455–61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBennett MJ, Albert RH, Jez JM, et al.: Steroid recognition and regulation of hormone action: crystal structure of testosterone and NADP+ bound to 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase. Structure. 1997; 5(6): 799–812. PubMed Abstract \| Publisher Full Text\n\nAndreini C, Bertini I, Cavallaro G, et al.: Structural analysis of metal sites in proteins: non-heme iron sites as a case study. J Mol Biol. 2009; 388(2): 356–80. PubMed Abstract \| Publisher Full Text\n\nMobley DL, Dill KA: Binding of small-molecule ligands to proteins: “what you see” is not always “what you get”. Structure. 2009; 17(4): 489–98. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrüger DM, Gohlke H: DrugScorePPI webserver: fast and accurate in silico alanine scanning for scoring protein-protein interactions. Nucleic Acids Res. 2010; 38(Web Server issue): W480–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnand P, Nagarajan D, Mukherjee S, et al.: ABS-Scan. Zenodo. 2014. Data Source" }	[ { "id": "6262", "date": "30 Sep 2014", "name": "Sunando Datta", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript 'ABS–Scan: In silico alanine scanning mutagenesis for binding site residues in protein–ligand complex' reports development of a web server for performing in silico alanine scanning mutations for studying protein-small molecule interactions. It further validates the tool by taking a list of already published Alanine scanning data along with the X-ray crystallographic structures of the relevant protein-ligand complexes. ABS-Scan provides a user-friendly web interface and will be very much useful for experimentalists to assess the outcome of mutations designed to study protein-ligand (small molecule) interactions. Overall the web tool is well explained in the manuscript.My main concern is the method for energy calculations, authors used to predict the individual contribution upon mutation. It does not include waters and metals. As it is well established that many of the protein-ligand interactions are water mediated and therefore water plays very important role in determining the specificity as well as affinity. Authors could use energy function which includes waters and metals as well otherwise the current version could only be used for protein-ligand complexes in which water/metal atoms have been shown to play any role in stabilizing the ligand in the binding pocket.Thus in my opinion, I think it could be indexed with inclusion of an updated energy function. Alternatively, its sole applicability for protein-ligand interaction without involvement of solvent molecules should be mentioned in the conclusion.", "responses": [ { "c_id": "1087", "date": "18 Nov 2014", "name": "Nagasuma Chandra", "role": "Author Response", "response": "We thank the reviewer for going through our manuscript and finding the work useful. These are indeed valid points and have been addressed in the revised version. Bridge water molecules do play an important role in the protein-ligand interactions. One has to take into account the resolution of the protein structure to determine the confidence of the placed water molecules. Hence an advanced option is provided wherein these water molecules when present at the site can be considered to be a part of the corresponding ligand moiety. The user can upload his/her own pdbqt file for the ligand with the appropriate water molecules added to it. An example of protein lysine methyltransferases complexed with S-adenosyl methionine has been described in the manuscript. The corresponding pqbqt file of the ligand can be downloaded from the example section. The results of these can also be accessed from example section of the web-server." } ] }, { "id": "6276", "date": "17 Oct 2014", "name": "Bernard Offmann", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper reports an attempt to develop an original tool that simulates alanine scanning mutagenesis to probe residues involved in the process of ligand recognition in proteins.More precisely, the work describes the development of a work flow that implements known methodologies for homology modeling of alanine single-point mutants of a protein and for molecular docking. Even though, this can be viewed as a methodological paper.We have some serious concerns regarding this work.The authors claim that they performed a \"validation\" of their tool on a dataset that comprises \"79 entries\" carefully selected from PDB (also cf point 2 below). Their evaluation is based on finding a correlation between docking scores with experimentally determined binding affinities. In their paper, the authors provide evidence of this validation by providing results of \"Experimental correlation\" for only one example (Figure 2) which relates to binding of rat 3-alpha-hydroxysteroid dehydrogenase (PDB: 1AFS) to testosterone and progesterone. Since they must have it, clearly, the authors should provide their evaluation of this correlation on all \"79 entries\". I would expect at least that they provide a new Figure 2 that comprises all data points coming from these \"79 entries\" to sustain their claim and help readers to evaluate the global performance of their tool. They attempted to provide few additional results on their website (http://proline.biochem.iisc.ernet.in/abscan/validation). It is more confusing because the results provided for the vitamin D receptor (PDB: 1IE9) is not about binding affinities but \"translational activity\". I'm here suggesting that detailed data for all mutations taken from all \"79\" entries are provided to the community in the form of a table or downloadable flat or excel-type file. The amount of independent PDB entries in their dataset is not 79. In fact, in some of PDB entries, multiple ligands were observed. Surprisingly, they consider these as separate entries. So their data is redundant with respect to the proteins. When generating homology models for protein variants, even if these are single point mutants, assessment of the quality of the models is a critical step. Selecting best models may not be that trivial. The authors need to clarify how they implement in their work flow the assessment of the quality of the models and consequently, what criteria they used for selecting the best models (and how many of them) that will be subjected to molecular docking. Regarding the alanine scanning procedure, there are issues regarding the treatment of alanine and proline. They should both be discarded from the alanine scanning protocol: alanine is already present in the structure while proline is not suitable for mutations because of the major protein backbone rearrangements that should be performed to properly mutate it. For such a tool, it is at stake to evaluate its performance using different homology modeling and molecular docking methods. The rational behind the choice of Modeler over other methods like Rosetta is not indicated. Likewise, the reason why Autodock and not Dock etc or even Autodock Vina is not explained. The efficiency of molecular docking using AutoDock is also dependent on the docking protocol used. In such an automated \"screen\", care should be taken about the preparation of the receptor, the ligand and the grid. For example, are the ligands kept flexible ? In the manuscript, there are no indications about how the authors dealt with this central issue. The authors are encouraged to describe precisely and discuss their docking protocol. According to the AutoDock 4.0 article, the median error range in energy estimation for any protein-ligand evaluation is 1.5-2.0 kcal/mol. In their study, the ∆∆G differences for ligand binding between mutant and native forms of the proteins are far below 2.0 kcal/mol. Thus, it is difficult to rank the mutants. Also, how the authors chose the 0.5 kcal/mol ∆∆G threshold is not clear. There is no discussion how this threshold compares with the intrinsic limits in precision of AutoDock. The definition of ligand in the tool is problematic. In case of oligo or polysaccharides, the carbohydrate residues are erroneously considered separately. For example, in the 1J84 entry from PDB, the carbohydrate-binding module (CBM) is bound to cellotretraose, a 1,4-β-D-glucan composed of four ß-D-glucose residues linked by ß-1,4 osidic linkages. When this PDB entry is submitted to ABS-Scan, it erroneously splits the oligomer into smaller entities that correspond to the chemical IDs of its constituents (BGC 401, 402, 403, 404). This is a serious flaw in their software. While it is common to see people to reuse available codes, the authors do not properly cite the source of their codes they posted on Github and used for providing a complete service to the community: at least 80% of the “alanine_scanning.py” code comes from either MODELLER examples (http://salilab.org/MODELLER/wiki/Mutate_model) or AutoDock code (http://mgltools.scripps.edu/api/AutoDockTools/AutoDockTools.Utilities24.compute_AutoDock41_score-pysrc.html).", "responses": [ { "c_id": "1086", "date": "18 Nov 2014", "name": "Nagasuma Chandra", "role": "Author Response", "response": "We thank the reviewers for their time and effort. There were some useful suggestions, which we have incorporated but do not agree with all the points raised. A detailed point-by-point response is given below.The authors claim that they performed a \"validation\" of their tool on a dataset that comprises \"79 entries\" carefully selected from PDB (also cf point 2 below). Their evaluation is based on finding a correlation between docking scores with experimentally determined binding affinities. In their paper, the authors provide evidence of this validation by providing results of \"Experimental correlation\" for only one example (Figure 2)which relates to binding of rat 3-alpha-hydroxysteroid dehydrogenase (PDB: 1AFS) to testosterone and progesterone. Since they must have it, clearly, the authors should provide their evaluation of this correlation on all \"79 entries\". I would expect at least that they provide a new Figure 2 that comprises all data points coming from these \"79 entries\" to sustain their claim and help readers to evaluate the global performance of their tool. They attempted to provide few additional results on their website (http://proline.biochem.iisc.ernet.in/abscan/validation). It is more confusing because the results provided for the vitamin D receptor (PDB: 1IE9) is not about binding affinities but \"translational activity\". I'm here suggesting that detailed data for all mutations taken from all \"79\" entries are provided to the community in the form of a table or downloadable flat or excel-type file.A suitable dataset for validation would be one that reports binding affinities for both wild-type and mutant proteins with same ligand, performed in a uniform experimental environment, for large number of proteins. Although such a dataset exists for protein-protein alanine scanning mutagenesis for eg., Rosetta alanine scanning), there are none reported for protein-ligand interactions.Since no such dataset was available to us, we systematically extracted PDB entries of ligand bound complexes and the corresponding binding sites in them that contained information about experimental alanine-scanning mutagenesis. However, the manner in which the effects of mutagenesis are reported in these differ significantly. While differences in ligand binding strengths (Ka or Kd values) are reported for some, changes in catalytic efficiencies are reported for some others. For some others, reporter assays are given which indicate capability of the downstream process more qualitatively. Hence it is difficult to perform a systematic comparison from these with the ∆∆G values calculated from our tool in this study. Nevertheless from this dataset, some examples were hand-picked, corresponding primary literature were read and known residue importances obtained, which were then compared with the predicted ones from our tool. In any case, ABS-Scan analysis has been successfully performed on 54 (the remaining 25 cases were not processed by default steps due to unusual atom types in proteins/ligands) complexes, which provide the extent of contribution to ligand binding of each residue in each site, in the form of a ranked list of residue-wide ∆∆G values. All this information has been made available to the community, through our webserver - (http://proline.biochem.iisc.ernet.in/abscan/validation).Besides this, given the lack of systematic reports of experimental data, validation can only be performed to understand the significance of the ∆∆G scores calculated from our tool. For this, we have taken two large datasets (a) protein complexes with native ligands versus decoy ligands from and (b) list of well curated with precise binding site definitions for known protein-ligand complexes used for benchmarking docking algorithms. From both of these, ∆∆G scores are in the range of 0.5 was significant.a) A fresh dataset derived from PDB-Bind core dataset consisting of 195 protein-ligand complexes, which has been developed for the purposes of benchmarking docking algorithms (Kim et al., 2004, Huang et al., 2008). Of the 195, 135 could be processed successfully for preparation of the protein-ligand complexes for analysis. (The others that could not be included, are likely to contain either unusual atom names or types or missing protein/ligand atoms or unusual convention for ligand atoms and hence could not be processed). b) A dataset of 343 protein-ligand complexes, each with a native and a decoy ligand. 288 structures out of 343 could be successfully evaluated. (Here again the others were omitted due to difficulties in automatic protein/ligand preparation).In the process, since ABSscan has been run for all these complexes, information about key contributing residues is generated for each of them. This has been made available through the webserver. Residue-wise contribution is obtained and presented in a ranked order for each complex, thus providing a ready resource of important residues for ligand binding.The results of these can be accessed from the validation section on the webserver – http://proline.biochem.iisc.ernet.in/abscan/validation The amount of independent PDB entries in their dataset is not 79. In fact, in some of PDB entries, multiple ligands were observed. Surprisingly, they consider these as separate entries. So their data is redundant with respect to the proteins. These reflect independent binding sites (with bound ligands). As can be expected, some proteins have multiple sites with different ligands, making it necessary to consider them separately. Hence 79 sites are unique and come from 46 PDB entries. In the original manuscript, the dataset of 79 was never meant to reflect ‘unique PDB entries’. In any case we refer to them now as ‘binding site entities’ to reflect this more clearly. When generating homology models for protein variants, even if these are single point mutants, assessment of the quality of the models is a critical step. Selecting best models may not be that trivial. The authors need to clarify how they implement in their work flow the assessment of the quality of the models and consequently, what criteria they used for selecting the best models (and how many of them) that will be subjected to molecular docking.Model quality has been considered as part of the modelling pipeline itself. Given the scale of the study, it is practical to generate one model for each mutant, but care is taken to ensure that it is optimal and free of errors in terms of bad contacts or atomic clashes. The optimization protocol used consists of 200 iterations of conjugate gradient, followed by molecular dynamic simulation for 4fs and simulated annealing with 200 iterations at different temperatures (This is the default protocol suggested in Model_mutate.py of Modeller - http://salilab.org/modeller/wiki/Mutate%20model). The initial restraints for generation of the model is derived from the wild-type structure itself. Assumptions necessary for modelling point mutations introduced through alanine-scanning mutagenesis protocol at the binding sites are that (a) they are unlikely to change the overall structure of the protein drastically and (b) the ligand moiety roughly retains the same conformation in comparison with the wild-type complex to interact with the mutated structure.Since modelling protocols have been well established for a long time now, we did not see the need for adding this information explicitly in the original MS. In any case, based on the reviewers suggestion, this information has been added to the revised version. Normalized DOPE scores are reported for both the native and mutant structures. DOPE refers to ‘Discrete Optimized Protein Energy’ and is a statistical potential which checks for the feasibility of the observed interactions. Protein structures with lower DOPE scores (typically in negative range -1.5 to -2.5 for experimentally solved structures) can be considered to be of good quality (Shen and Sali., 2006). Regarding the alanine scanning procedure, there are issues regarding the treatment of alanine and proline. They should both be discarded from the alanine scanning protocol: alanine is already present in the structure while proline is not suitable for mutations because of the major protein backbone rearrangements that should be performed to properly mutate it.This required addition of simple screens to filter out these residues from consideration for alanine scanning, which has been done. Changes have been made to both the source code and the web-tool now. Glycine mutations are also filtered out. For such a tool, it is at stake to evaluate its performance using different homology modeling and molecular docking methods. The rational behind the choice of Modeler over other methods like Rosetta is not indicated. Likewise, the reason why Autodock and not Dock etc or even Autodock Vina is not explained.The goal of our study is not to develop a modelling algorithm or a new parameter for building models. The most widely used tool for homology modelling – Modeller, which we have currently included in the workflow, has about 1500 citations. Currently there are more than 50 tools for homology modeling -(http://en.wikipedia.org/wiki/List_of_protein_structure_prediction_software) and roughly the same number of tools for protein-ligand docking (http://en.wikipedia.org/wiki/Docking_%28molecular%29). The precise reason for choosing ‘Modeller’ or ‘Autodock’ is perhaps because of our own experience in using these tools along with availability of extensive documentation, tutorials and ease of implementation. Moreover, both these libraries had python bindings available and hence could be merged into a single script using python. In future, we plan to develop a pymol plugin for the same. A simple bash script for processing the protein-ligand complex to determine the interaction energy using ROSETTA force fields has also been included in the github repository. This again, is only for the advanced users and we might incorporate it in the future versions of the pipeline. The efficiency of molecular docking using AutoDock is also dependent on the docking protocol used. In such an automated \"screen\", care should be taken about the preparation of the receptor, the ligand and the grid. For example, are the ligands kept flexible? In the manuscript, there are no indications about how the authors dealt with this central issue. The authors are encouraged to describe precisely and discuss their docking protocol.We would like to clarify here that there is no docking performed in the whole exercise. We only score the complex in the given conformation using the force fields. By default, through prepare_receptor4.py and prepare_ligand4.py Gasteiger charges and polar hydrogens are added while evaluating the interaction energy. This has been mentioned in the manuscript:“Each mutated structure, will then be scored by using Autodock 4.1 force field, to calculate the energetics of a protein-ligand complex. The contribution from the residue is then determined by calculating the difference in interaction score of the mutant and the wild-type protein (∆∆G value).” According to the AutoDock 4.0 article, the median error range in energy estimation for any protein-ligand evaluation is 1.5-2.0 kcal/mol. In their study, the ∆∆G differences for ligand binding between mutant and native forms of the proteins are far below 2.0 kcal/mol. Thus, it is difficult to rank the mutants. Also, how the authors chose the 0.5 kcal/mol ∆∆G threshold is not clear. There is no discussion how this threshold compares with the intrinsic limits in precision of AutoDock.The median error range of the energy estimation reported in AutoDock 4.0 article is for the total ∆G score between the experimental and predicted values, whereas in this case it is for individual residue contributions. The distribution of the ∆∆G values obtained for the decoy and cognate ligands from the CSAR dataset (http://www.csardock.org/) was used to define a cut-off of 0.5. This has also been validated on PDBbind core dataset (http://www.pdbbind-cn.org/). Figures 3A and 3B have been added along with explanations in the manuscript.We believe that intrinsic limits on precision of Autodock scoring would not be a major concern as both the wild type and the mutant are evaluated using the same scoring scheme and the cut-off has been chosen on basis of native protein-ligand complexes in CSAR and PDBbind datasets. The definition of ligand in the tool is problematic. In case of oligo or polysaccharides, the carbohydrate residues are erroneously considered separately. For example, in the 1J84 entry from PDB, the carbohydrate-binding module (CBM) is bound to cellotretraose, a 1,4-β-D-glucan composed of four ß-D-glucose residues linked by ß-1,4 osidic linkages. When this PDB entry is submitted to ABS-Scan, it erroneously splits the oligomer into smaller entities that correspond to the chemical IDs of its constituents (BGC 401, 402, 403, 404). This is a serious flaw in their software.This is not really a 'problem' and is an established work-around to avoid long computation and hence long waiting time for the user. All this does is to split peptides or oligosaccharides into individual moieties (typically for a peptide, each amino acid is considered as a moiety and for an oligosaccharide, each monosaccharide is considered as a moiety), as per the convention currently followed by PDB. How can this be a ‘serious flaw’? It does not, in any manner, influence the results. Many other tools for protein-ligand interaction analysis such as LPC (Ligand-protein contacts, Ligplot+, Ligplus etc.) also track ligands through such residue identifiers.However, an advanced option has now been added to provide the range of the ligand residue numbers to be considered as a single moiety during the entire protocol. For example, now a residue range 401-404 can be provided for 1J84 instead of a single residue number to consider the whole oligocomplex as single ligand. The script has also been accordingly modified in github. While it is common to see people to reuse available codes, the authors do not properly cite the source of their codes they posted on Github and used for providing a complete service to the community: at least 80% of the “alanine_scanning.py” code comes from either MODELLER examples (http://salilab.org/MODELLER/wiki/Mutate_model) or AutoDock code (http://mgltools.scripps.edu/api/AutoDockTools/AutoDockTools.Utilities24.compute_AutoDock41_score-pysrc.html).We have indeed already cited all the tools used in the manuscript to which source codes are linked. In any case, these references are now highlighted in the source code also. Both Autodock and Modeller are released under the GNU public license, making their source code freely usable to all interested parties. Moreover, these are the primary source and codes are not extracted from any third-party tools. The purpose of putting it on Github is to be completely open about the details of the protocol and make our work fully accessible to anyone interested. We would again like to remind the reviewer here that source-code is used only by an advanced user. The reviewer may be aware of the time and effort involved in producing a web-application interface that is embedded with visualization features. This has been done with the belief that it will save precious time for researchers who do not have the expertise or the interest in installation and handling command-line interfaces for such tools. We initially proposed this as a web-tool, but since that section is no longer available in F1000Research, we submitted it as a software tool." } ] } ]	1	https://f1000research.com/articles/3-214
https://f1000research.com/articles/3-287/v1	21 Nov 14	{ "type": "Software Tool Article", "title": "Selecting relevant nodes and structures in biological networks. BiNAT: a new plugin for Cytoscape", "authors": [ "Fabio Cumbo", "Giovanni Felici", "Paola Bertolazzi", "Giovanni Felici", "Paola Bertolazzi" ], "abstract": "Summary: In order to understand a network function, it’s necessary the understanding of its topology, since the topology is designed to better undertake the function, and the efficiency of network function is influenced by its topology. For this reason, topological analysis of complex networks has been an intensely researched area in the last decade.Results: Here we propose BiNAT, a Cytoscape [1] plugin able to perform network analysis, providing a full set of useful tools to discover the most significant nodes and structures in a network.Conclusions: The plugin has been approved on the official Cytoscape plugins repository and it is downloadable directly from this site: http://dmb.iasi.cnr.it/binat.php where a full guide is also available.", "keywords": [ "graph theory", "biological networks", "network analysis", "cytoscape plugin" ], "content": "Background\n\nIn this section we briefly introduce the fundamental concepts that are needed to understand the principles of the software described in this article. We introduce the main theoretical concepts used to describe and analyze networks, most of which come from graph theory that is a large field containing many results and we describe only a small fraction of those here, focusing on the ones that are relevant to the study of real-world networks.\n\nTo represent a graph there are many different ways in literature. A graph is usually represented as an adjacency matrix. The adjacency matrix A of a simple graph is the matrix with elements aij, where aij = 1 if an edge between vertices i and j is present, and 0 otherwise. We note that a) for a graph with no self-edges the elements in the matrix diagonal are all zero, b) the adjacency matrix is symmetric (if there is an edge between i and j then there is an edge between j and i). In some situations it is useful to associate a weight to the edges; such weighted (or valued) graphs can be represented by giving the elements values of the adjacency matrix equal to the weights of the corresponding connections.\n\nNetwork centrality measures allow categorizing nodes for their relevance in the network structure. The literature on biological networks are typically interested in the global properties of the network; in the case of centrality it is often considered from a global point of view, as for example analyzing degree or centralities distribution2–6. In BiNAT were implemented the most used network centrality measures i.e. Degree, Betweenness, Eccentricity, Closeness and Stress centrality.\n\n\nSoftware overview\n\nThe increasing availability of large network datasets, along with the progress in experimental high-throughput technologies, have promoted the need for tools that allow an easy integration of experimental data with data derived from network computational analysis. In order to enrich experimental data with network topological parameters, it has been developed the Cytoscape plugin BiNAT (Biological Network Analysis Tool).\n\nThe plugin computes several network centrality parameters, and allows the user to analyze these computational results in textual and graphical format. BiNAT identifies the nodes that are relevant from the experimental and the topological viewpoint. BiNAT is one of the few Cytoscape plugins that computes several centrality indices at once. In BiNAT the centrality measures can be easily correlated with each other, in order to identify the most significant nodes according to topological properties. Functional to this capability is the scatter plot options, which allows an easy correlation of node centralities.\n\nEqually to other Cytoscape plugins, BiNAT (and its dependencies) must reside in the plugin directory in the Cytoscape root folder. BiNAT needs to be executed with administrative privileges to working proper. After first running, BiNAT creates three folders (binatData, binatLog and binatTemp) in the Cytoscape root directory:\n\nin binatLog folder application log files reside in two formats (.html and .txt), both useful to monitor the proper running of all software operations.\n\nin binatData folder generated application data reside such as: a) stats.xlsx file that contains network information (centrality measures for every nodes, network diameter, network density, node average degree, network maximal clique degree, etc). b) complexesIntersectionMatrix.xlsx file that represents the complexes adjacency matrix computed according to the number of shared nodes between complexes. c) complexesShortestPathsMatrix.xlsx file that represents the complexes adjacency matrix computed according to the shortest path among all shortest paths between two nodes that are members of the same complex. d) shortestPaths.txt file that contains all pairs shortest paths in the network, useful to accelerate many other plugin features.\n\nin binatTemp some plugin temporary files reside such as: a) sp_.txt files that contain all pairs shortest paths in the network. The maximal size of every sp_.txt file is 50MB and their creation may take a long time depending on the network size. b) toResume.txt file that contains two indexes necessary to resume the process of calculating shortest paths if it has been stopped.\n\n\nImplementation\n\nThe idea behind the developed interface consists of a clear separation from the visualization of results and the Java classes that contain the processing logic. The structure is comparable to the MVC (Model - View - Controller) design pattern that is based on the separation of roles between the software components that interpret three major roles:\n\nModel: application data and rules.\n\nView: it can be any output representation of data, such as a chart or a diagram.\n\nController: it mediates input, converting it to commands for the model or view.\n\nThe Controller is the key class that guides the input information flow (commands and parameters) towards the right actions to be performed.\n\n\nImplemented algorithms\n\nIn this section the algorithms implemented in BiNAT are introduced through a pseudocode and a comprehensive description for each algorithms. The following algorithms have been implemented: Dijkstra algorithm, Betweenness centrality, Closeness centrality, Degree centrality, Eccentricity centrality, Stress centrality and Clique Finder (Bron-Kerbosch) algorithm.\n\nDijkstra’s algorithm7 solves the single-source shortest path problem when all edges have non-negative weights. It is a greedy algorithm, similar to Prim’s algorithm, but the two solve different types of problems and the properties are computed in different ways. Algorithm starts at the source vertex s and grows a tree T that ultimately spans all vertices reachable from s. Vertices are added to T in order of distance i.e., first s, then the vertex closest to s, then the next closest, and so on. The following implementation (see Listing 1) assumes that the graph G is represented by adjacency lists. As already mentioned, all centrality measures available in BiNAT, excluded the Degree centrality, depend on the Dijkstra’s shortest path algorithm.\n\nListing 1. Pseudocode of the Dijkstra’s shortest path algorithm.\n\n\n\nThe Clique Finder algorithm that we use was developed by Bron & Kerbosch (1973)8. This algorithm combines a recursive backtracking procedure with a branch and bound technique to eliminate searches that cannot lead to a clique. The recursive procedure is self-referential: finding a clique of length n is accomplished by finding a clique of length n-1 and another node that is connected to all the nodes in that clique. The branch and bound technique makes use of rules that allow us to determine in advance certain cases for which possible combinations of nodes and edges will never lead to a clique. There are three sets that are essential for this algorithm:\n\n“potential-clique”: this is a set of nodes where every node is connected to every other node. Each recursive call will either extend this set by one node or reduce it by one node.\n\n“candidates”: this is the set of nodes that are eligible for addition to the “potential-clique” set.\n\n“already-found”: this is the set of nodes that have already served as an extension to the present configuration of “potential-clique” and are now explicitly excluded. That is, all possible extensions of “potential-clique” containing any point in this set have already been generated.\n\nThe algorithm operates recursively on each of the sets by generating all extensions of a given configuration of “potential-clique” that use given set of “candidates” and that do not contain any of the nodes in “already-found”, as described in the simplified pseudocode represented in Listing 2. Initially, the set “candidates” contains all the nodes in the graph and the set of “potential-clique” and “already-found” are empty. Bron & Kerbosch adopt a clever strategy to select the nodes: to choose nodes with the largest number of edges, in order to reach the branch and bound condition as soon as possible. This leads to the larger cliques being found first and sequentially generates cliques having a large common intersection. More details of this algorithm, including a more detailed pseudocode, implementation are given by8.\n\nListing 2. Pseudocode of the Bron-Kerbosch algorithm.\n\n\n\n\nSupported features\n\nOne of the most important commands in BiNAT is the one for the creation of the principal output file (in MS Excel or CSV format), in which all network nodes with all their own centrality measures defined before are listed: the ranking measure has been introduced to find a correlation between centrality measures that are in conflict with each other; it is a value ranging from 0 to 10, where highest value are received by nodes that are candidates to be considered hubs. This file creation step passes through almost all stages of operational calculus which constitute the heart of the software. The first step consists is the input of a network. The network must be in TXT format according to the TAB2 standard. The format supported until now is that adopted by BioGRID. To compute all nodes and network centrality measures, it is needed to type the “makestatistics” command in the plugin text field (please see the software manual on the official site for more information about available commands). BiNAT will then performs the following steps:\n\nFirst of all BiNAT computes all pairs shortest paths in the network using Dijkstra algorithm7 (it is needed to compute almost all centrality measures);\n\nOnce obtained all shortest paths, BiNAT computes all supported centrality measures for each node in the network.\n\nThen BiNAT assigns a ranking value to each node; such ranking is obtained calculated as the arithmetic average value of all centrality measure for each node. Once completed, all centrality measures for all nodes in the network, BiNAT computes other global measures such as the network density, the network diamenter, the maximal clique degree and much more.\n\nData is ready to be returned in output now. BiNAT creates the output file in the folder specified at the beginning of the operation. To create XLSX files, BiNAT uses the Apache POI libraries: Java API for Microsoft Documents. The aim of the Java POI is to manipulate various file format based upon the Office Open XML standards (OOXML) and Microsoft’s OLE 2 Compound Document format (OLE2). In short, the programmer is able to read and write MS Excel, MS Word and MS PowerPoint files using Java.\n\nFor a list of all supported command line options, please take a look at the User Manual on the plugin official site. BiNAT also provides a server mode that allows the user to fully remotely control the plugin. When the server mode is enabled, user can send commands remotely using BiNAT client available as desktop and mobile application (for Android devices only).\n\n\nBiNAT at work\n\nWe tested BiNAT on Saccharomyces Cerevisiae (Figure 1) protein-protein interaction network. PPI network was extracted from BioGRID database and consist of 6,096 nodes and 214,235 interactions.\n\nIt was analyzed with information about yeast complexes provided by the Wodak Lab (Molecular Structure & Function program, Hospital for Sick Children, Toronto, ON, Canada). We first used BiNAT to compute all the centrality measures. A first overview of the global topological properties comes from the values of all centralities along with the diameter and the nodes average degree (Figure 2).\n\nWith this data and with the help of a graphical layout, we can deduce a highly connected network. The computation of network centrality measures allow us a first ranking of this network. Further analysis bases on a Gene Ontology database search, or by adding functional annotation data, may allow a deeper functional exploration of the network. The combination of BiNAT with other bioinformatics tools, such as CentiScaPe or Network Analyzer or MCODE, may help to analyze high-throughput genomic and or proteomic experimental data and facilitate the analysis process. It’s recommended to see the plugin User Manual for a step-by-step guide on how to use BiNAT and all its features.\n\n\nConclusions\n\nThe Cytoscape plugin BiNAT was designed to provide the user a powerful tool for an accurate analysis of networks centrality. The plugin interface is simple as a shell; a practical user manual can be downloaded from the official web site http://dmb.iasi.cnr.it/binat.php.\n\nBiNAT plugin has been accepted by the Cytoscape community and is actually available for the download on the official plugins repository for the 2.8.3 version of Cytoscape.\n\n\nSoftware availability\n\nSoftware available from Cytoscape Plugins repository: http://chianti.ucsd.edu/cyto_web/plugins/ and search for binat\n\nhttp://dmb.iasi.cnr.it/binat.php\n\nhttp://dx.doi.org/10.5072/zenodo.127719\n\nGNU General Public License v2.0", "appendix": "Author contributions\n\n\n\nPB conceived research. FC developed software. FC, PB and GF analyzed data and results. All authors wrote the paper.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nFC thanks financial support from the National Research Council of Italy (CNR).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nShannon P, Markiel A, Ozier O, et al.: Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003; 13(11): 2498–2504. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJeong H, Tombor B, Albert R, et al.: The large-scale organization of metabolic networks. Nature. 2000; 407(6804): 651–654. PubMed Abstract \| Publisher Full Text\n\nJoy MP, Brock A, Ingber DE, et al.: High-betweenness proteins in the yeast protein interaction network. J Biomed Biotechnol. 2005; 2005(2): 96–103. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWagner A, Fell DA: The small world inside large metabolic networks. Proc Biol Sci. 2001; 268(1478): 1803–1810. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWuchty S, Stadler PF: Centers of complex networks. J Theor Biol 2003; 223(1): 45–53. PubMed Abstract \| Publisher Full Text\n\nYamada T, Bork P: Evolution of biomolecular networks: lessons from metabolic and protein interactions. Nat Rev Mol Cell Biol. 2009; 10(11): 791–803. PubMed Abstract \| Publisher Full Text\n\nDijkstra EW: A note on two problems in connexion with graphs. Numerische Mathematik. 1959; 1(1): 269–271. Publisher Full Text\n\nBron C, Kerbosch J: Algorithm 457: finding all cliques of an undirected graph. Commun ACM. 1973; 16(9): 575–577. Publisher Full Text\n\nCumbo F, Felici G, Bertolazzi P: BiNAT: new plugin for Cytoscape. Zenodo. 2014. Data Source" }	[ { "id": "6765", "date": "04 Dec 2014", "name": "Emidio Capriotti", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper briefly describes BINAT, a new Cytoscape plugin to calculate several network features. The plugin provides an interface to export the results of the calculation in an excel file.The authors should have better described the advantages of using their plugin showing, for example, the computing time required to perform calculation on networks with different size.", "responses": [] }, { "id": "7310", "date": "21 Jan 2015", "name": "Hagen Blankenburg", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript presents BiNAT, a Cytoscape plugin for computing a number of network centrality parameters. In the last years, topological network analyses have yielded interesting insights, thus this is an important area of research. However, I see a number of major issues with both the software and the accompanying manuscript that should be addressed. - Similar plugins:As the authors state, “BiNAT is one of the few Cytoscape plugins that computes several centrality indices at once.” The other tools should not just be named at the end of the manuscript but should be properly cited and described in the introduction. NetworkAnalyzer is included as a core plugin in every Cytoscape installation; CentiScaPe is available via the Cytoscape App store and has been published in F1000Research as part of the Cytoscape App collection. Although novelty is not a criterion for publication in F1000Research, without a thorough comparison to these tools it is hard to tell what BiNAT can do that these tools cannot (e.g. if there are particular centrality measures that only BiNAT can compute). - Cytoscape 2/3 installation procedure:I had some difficulties trying to install BiNAT. CentiScaPe can be installed with a single click in the Cytoscape App store; why is this elegant option not available for BiNAT? I could only test BiNAT with the outdated Cytoscape 2.8, as in Cytoscape 3.2 for MacOS I could not find a “plugin” folder (and creating the folder with the BiNAT files inside did not have an effect). This made me wonder if BiNAT is only compatible with Cytoscape 2 and not with the recent Cytoscape 3 (the compatibility entry in the plugin directory and the missing entry in the App store suggest this)? While of course I would like to see compatibility with Cytoscape 3, it should at least be clearly stated if this is not the case in order to prevent confusion. - Usability / user interface:While the authors state that \"[t]he plugin interface is simple as a shell\", a user that is familiar with the intuitive, few click interfaces of NetworkAnalyzer and CentiScaPe might find BiNAT’s command line interface quite challenging and unintuitive. If there is a clear benefit of this command line over a graphical and clickable interface this should be described with good examples (e.g. does it allow to script and save a complex analysis?). As a minimum example, the commands required to compute the Yeast network mentioned in the manuscript could be provided. However, for the user that does not feel overly comfortable typing commands into a shell (and I assume that Cytoscape is used by quite a few of those), a graphical, clickable, and intuitive interface to BiNAT seems to be essential. - File formats:Why does BiNAT actually need the ability to read network files? Input file handling and network creation should be completely handled by Cytoscape, which has a general table import function that can handle any kind of plain text file, not just BioGRID Tab 2.0; plugins like BiNAT should work on the final networks and not replicated core functionality. - Manuscript:In its current form the manuscript contains too many technical descriptions. Implementation details like the model-view-controller design pattern, the description of BiNAT’s internal directory structure and file organization, or what Apache module is used for writing Microsoft Excel files might be interesting for a developer but are irrelevant for the reader that is a potential user. All this text could be moved to the author’s website, freeing plenty of space for a comparison to the other available network analysis tools and for practical use-cases and examples. In a final step, a language check should be performed to make sure the text is fully comprehensible. - Figures:Figure 1 does currently not contain much relevant information and should be replace with a figure that is somehow connected to BiNAT. A classic approach would be to use the computed centrality parameters in Cytoscape’s VizMapper to change the color/size of important nodes, ideally focusing on a subnetwork of the Yeast interactome. The Cytoscape Tumblr might work as a great source of inspiration for useful and aesthetically pleasing Cytoscape representations. In addition, Figure 2 should probably be Table 1 instead, as it only contains tabular text.", "responses": [] }, { "id": "7588", "date": "18 Feb 2015", "name": "Vivek Anantharaman", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI reviewed this plugin as an end-user and as a biologist. I have created and analyzed many networks using Cytoscape. I installed the BiNat plugin, but unfortunately was not able to test its functionality because of a flaw I encountered in the initial step. I was unable to load my networks on to the plugin.The author gives two formats that are compatible. My main problem with this is that I am already using cytoscape and I have uploaded a network on Cytoscape. I should be able to use the network I have already uploaded and working with, rather than loading it a second time on to the plugin. At the least it should take the same formats that I was able to load onto Cytoscape (which did not work) or the *.cys format that cytoscape saves. The \"import network from table\" feature of cytoscape takes tab delimited files. The plugin was unable to take the very same files. This is a major drawback and a fatal flaw in my opinion. The second issue is the English in the paper and the manual. It needs extensive correction and reworking to be legible.This may very well be a useful and effective plugin. But I was unable to test its functionality because of a) poor instructions in the paper/manual , b) inability in using existing cytoscape networks and formats (This is a major drawback, and the utility of a plugin which cannot act on a network loaded in the main cytoscape window or at least makes use of the same format is very limited).I recommend that the author rework these issues and make the plugin more robust.", "responses": [] } ]	1	https://f1000research.com/articles/3-287
https://f1000research.com/articles/3-284/v1	19 Nov 14	{ "type": "Case Report", "title": "Case Report: Neotendon regeneration and repair of gluteus tendon tear at 1-year follow-up after ultrasound guided platelet rich plasma tenotomy", "authors": [ "Arockia Doss" ], "abstract": "Greater trochanteric pain syndrome (GTPS) is a common condition resulting in posterolateral hip pain typically in perimenopausal women. Gluteal tendinopathy is the underlying pathology and contributes to health care cost burden as a poorly managed tendon disorder. There is no established effective treatment for gluteal tendon pathology in GTPS. This article describes clinical, imaging and life style improvements after percutaneous tendon repair using autologous platelet rich plasma tenotomy under ultrasound imaging guidance in a case of GTPS. The improvements observed in this patient add to the call for urgent medical and economical need for more research on percutaneous tendon repair.", "keywords": [ "GTPS", "gluteal tendinopathy", "tendon repair", "tenotomy", "ultrasound imaging" ], "content": "Introduction\n\nGreater trochanteric pain syndrome (GTPS) is a common cause of posterolateral hip pain typically seen in peri and post menopausal women. Coexistent obesity, ilio-tibial band (ITB) syndrome, low back pain, osteoarthritis are added risk factors1. Evaluation using imaging and histology techniques revealed degenerative tendinosis with tears of the gluteal tendons2–4. The primary underlying pathology of GTPS is a gluteal tendinopathy with or without tears of the tendon5. Although GTPS is widely referred to as greater trochanteric ‘bursitis’, there is no inflammation of the greater trochanteric bursa on histological evaluation of surgical specimens6.\n\nThe natural history of GTPS is favourable in most cases and responds to physical therapy, weight loss, non-steroidal anti-inflammatory drugs and behaviour modification1. However in some patients the condition causes significant disability and necessitates intervention. Corticosteroid injection into the bursa and surgical repair of any torn gluteal tendons are the current common treatment options. Corticosteroid injections are controversial in a degenerating tendinopathy7. Corticosteroid injections carry the risk of a dampening effect and progressive worsening of tendon pathology8. A pioneering percutaneous treatment for all tendoligamentous and cartilage tears using autologous platelet rich plasma (PRP) tenotomy under high resolution imaging control was routine clinical management in the author’s practice. The same treatment was performed in a patient with GTPS. This report is the 1-year follow up on the clinical outcome and imaging appearance of this patient.\n\n\nCase report\n\nA 56 year old female Caucasian patient presented with 6 months of progressive left sided greater trochanteric pain syndrome. She complained of pain every day for many months, was unable to climb stairs and experienced moderate stiffness of the outer hip during early morning awakening. Pre-procedural clinical examination revealed an overweight individual with a BMI of 27, valgus knees, gynacoid pelvis, localised tenderness of the gluteus minimus and medius insertions into the facets of the greater trochanter, painful limitation of passive and active hip abduction and provocation to resisted abduction due to gluteal tendon dysfunction. She wished to be very active, commence a walking holiday and reduce her body weight. She refused both corticosteroid injection therapy and surgery. Full written informed consent to treat and publish the data was obtained from the patient. Ultrasound scan showed a 10 mm × 12 mm high grade full thickness tear of the degenerating gluteus medius tendon insertion (Figure 1) and gluteus minimus tendinopathy with partial split tears (image not shown). Under ultrasound imaging (GE Logic 9, 9MHz probe) control, 1% lignocaine 5 cc was infiltrated through a 22 G needle into and around the minimus tendon. Percutaneous tenotomy was performed into the foot print and the adjacent gluteal cuff under real time imaging guidance. 4–5 cc of autologous PRP (REGEN Switzerland, Adistem Hong Kong) was infiltrated. Repeated percutaneous tenotomy with PRP of the medius tendon was performed in a similar manner 12 days later. Routine rehabilitation with range of motion exercises, graded activity and a strengthening program was commenced. She took no time off work. Her symptoms improved within 4 weeks. At 6 months post treatment, she enjoyed a walking holiday in the hilly terrains of the Kimberly, Western Australia without limitation. Clinical and ultrasound exam follow-up at 12 months revealed a weight reduction of 6 kg with a near normal BMI of 25.1. Her daily pain had resolved, with moderate pain only on ascending stairs. There was minimal greater trochanteric tenderness and no limitation of hip abduction. Ultrasound of the gluteal tendons revealed that neotendon tissue had replaced the degenerative tendinotic tissue with obliteration of the previously known tear defect (Figure 2).\n\n\nDiscussion\n\nTendon pathologies in an ageing population contribute to a significant bulk of musculoskeletal pain syndromes resulting in a major health burden worldwide. They represent the third highest health care expenditure, costing AUD$517 million per annum in Australia9. To date there are no effective treatment strategies that uniformly address the pathology of degeneration in tendinopathy7.\n\nFor those patients who do not respond to conservative measures, the current options for treating tendinopathy by corticosteroid injections are ineffective7. Corticosteroid injections may result in progression of a catabolic tendon environment with worsening of tendon degeneration, dampening of protective pain sensory feedback resulting in over-activity and tendon rupture. Surgical repair is reserved as the very last option and data on efficacy is limited to retrospective case series10–12. Data from 2008 suggest that direct surgical costs and indirect costs from bone and joint disorders amount to USD 915 billion in the United States13. This is likely to escalate in the next few decades13. Therefore there is a medical and economic need to treat tendon disorders with innovative methods that will address these issues. The ideal procedure should aim to repair the tendon with minimum direct and indirect expenditure, be repeatable in a single course of treatment, reproducible across health care systems and widely available.\n\nThe search for new options should begin with an understanding of the basic anatomy, physiology and pathophysiology of tendon disorders. Tendons do not possess adequate vascular or nerve supply14. The ability of the human body to repair tendons is therefore inherently limited. The paratendon structures surrounding tendon fibrils contain neurovascular tissue that enables neuronal mediation and reparative tendon homeostasis via immunomodulatory and inflammatory molecular pathways. Dysfunctional neuronal mediation and an inadequate tendon homeostasis are the causes of chronic tendon disorders. An understanding of this inadequacy is the key to treating chronic painful dysfunction in tendon disorders14.\n\nIt is intuitive to suggest that the initial step should aim for the correction of limitation of neurovascular supply of tendons. Such a correction may facilitate neuronal mediation and improve immunomodulatory and inflammatory molecular pathways. This may pave the way for an adequate, albeit prolonged normal healing response. On this basis, biologicals that possess an anti-inflammatory/immunomodulatory effect that promote neo-tissue regeneration and neo-angiogenesis may offer a solution. The optimal biological material should facilitate neuronal mediation and tendon homeostasis.\n\nThe second step is the understanding that the macrostructure of the musculotendinous junction, the full extent of the tendon and the enthesis at the tendon bone fibrocartilage interface are implicated in chronic tendinopathy. Therefore any treatment should be aimed at correction of the entire extent of this anatomy where possible. Delivery of therapeutic material into the affected tendon should fulfill the requirement of being able to penetrate 1) through splits and tears within the substance of the tendon, 2) surrounding paratendon and the 3) fibrocartilage footprint of the insertional portion of the tendon, ensuring that as much affected tendon is treated as possible.\n\nIn addition, there is regional attrition of more than one tendon in any given region and any treatment should address augmentation of these regional tendons to ensure additional ‘scaffolding’ of a progressively weakened attritional environment. Placement of therapy through the neighbouring attritional tissues may contribute to additional scaffolding via ‘neotissue’ regeneration around a weakened tendon environment. For example, in gluteal minimus/medius tears, the iliotibial band, gluteus maximus interface should be augmented. As far as the author is aware, this concept of percutaneous tendon augmentation via biologicals has not been previously described in the literature.\n\nAutologous PRP is a supra physiologic concentration of platelets containing various growth factors secreted by the alpha granules in platelets. PRP has gained increasing and controversial popularity in the past few years. Application of PRP in treating degenerative rotator cuff lesions is made on the basis of its role in the regulation of matrix gene expression and cell proliferation15. The application of PRP into the enthesis is based on the regenerative effects on meniscal cells that share similar fibrocartilage histology16. Previous reports of autologous PRP are mixed, with some showing improvement, failure of intervention and reflect the various preparations of therapeutic material and the mode of delivery of PRP usually as an injection17,18. The role of high resolution imaging control in the delivery of therapeutic material is either absent or unclear in these prior reports.\n\nThis case report is unique in the simultaneous correlation of clinical and imaging findings as part of routine clinical practice. This ensured an accurate diagnosis of the afflicted tendons and was followed by delivery of the percutaneous treatment precisely into the culprit tendon and the augmentation of surrounding tissues using high resolution imaging control. This procedure represents a unique delivery of care model where clinical evaluation, imaging confirmation and percutaneous treatment were all performed by a single treating specialist dedicated to image-guided orthopaedic intervention. On this basis, the results here shown are incomparable to other existing published studies that use a different service delivery model.\n\nIn this patient, there was a combination of clinical improvement together with imaging follow-up demonstrating neotissue tendon regeneration within the gluteus medius tendon tear. The reduction in body weight and the improved BMI are important markers for improved outcome in lower limb tendon disorders. This implies improved mobility and overall health due to life style modifications subsequent to the treatment.\n\nThe author has previously published data on neotissue regeneration in a full thickness rotator cuff tear with complete abolition of pain that lasted nearly two years post percutaneous repair in an elderly patient19. As far as the author is aware this is the second report on neotissue tendon regeneration following ultrasound guided percutaneous liquid PRP tenotomy and the first report of percutaneous repair of a gluteus medius tendon tear.\n\nThis single report cannot form the basis of routine clinical application in other dissimilar service delivery models. Clinical use of this technique should be subject to regular clinical follow-up and outcome evaluation. Further research is needed prior to routine clinical use outside these parameters.\n\nIn conclusion, the treatment of degenerative tendon tears has entered a new paradigm. In this patient, percutaneous tendon repair under imaging guidance with autologous PRP tenotomy resulted in neotissue tendon regeneration of a gluteal tendon tear. This correlated with improvement in the clinical syndrome, implementation of life style modification, reduced BMI with very minimal loss of revenue due to time off work. This suggests that percutaneous repair may efficiently address the issues of the pathology and help in minimizing a sedentary life style in an increasingly overweight population with very minimal or no loss of time from work. This in turn should reduce healthcare costs to a stretched health system, loss of revenue to the patient and the indirect costs to the community. There is an economical and medical need for more research on this new paradigm shift in tendon repair.\n\n\nConsent\n\nWritten informed consent for medical treatment and publication of this anonymized report was obtained from the patient.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this study.\n\n\nAcknowledgements\n\nThe author is grateful to his referring health professionals and patients.\n\n\nReferences\n\nWilliams BS, Cohen SP: Greater trochanteric pain syndrome: a review of anatomy, diagnosis and treatment. Anesth Analg. 2009; 108(5): 1662–70. PubMed Abstract \| Publisher Full Text\n\nKong A, Van der Vliet A, Zadow S: MRI and US of gluteal tendinopathy in greater trochanteric pain syndrome. Eur Radiol. 2007; 17(7): 1772–1783. PubMed Abstract \| Publisher Full Text\n\nLequesne M, Mathieu P, Vuillemin-Bodaghi V, et al.: Gluteal tendinopathy in refractory greater trochanter pain syndrome: diagnostic value of two clinical tests. Arthritis Rheum. 2008; 59(2): 241–246. PubMed Abstract \| Publisher Full Text\n\nRobertson WJ, Gardner MJ, Barker JU, et al.: Anatomy and dimensions of the gluteus medius tendon insertion. Arthroscopy. 2008; 24(2): 130–136. PubMed Abstract \| Publisher Full Text\n\nAlvarez-Nemegyei J, Canoso JJ: Evidence-based soft tissue rheumatology: III: trochanteric bursitis. J Clin Rheumatol. 2004; 10(3): 123–4. PubMed Abstract \| Publisher Full Text\n\nSilva F, Adams T, Feinstein J, et al.: Trochanteric bursitis: refuting the myth of inflammation. J Clin Rheumatol. 2008; 14(2): 82–6. PubMed Abstract \| Publisher Full Text\n\nCoombes BK, Bisset L, Vicenzino B: Efficacy and safety of corticosteroid injections and other injections for management of tendinopathy: a systematic review of randomised controlled trials. Lancet. 2010; 376(9754): 1751–67. PubMed Abstract \| Publisher Full Text\n\nHart L: Corticosteroid and other injections in the management of tendinopathies: a review. Clin J Sport Med. 2011; 21(6): 540–1. PubMed Abstract \| Publisher Full Text\n\nhttp://www.aihw.gov.au/WorkArea/DownloadAsset.aspx?id=6442457279.\n\nDavies JF, Stiehl JB, Davies JA, et al.: Surgical treatment of hip abductor tendon tears. J Bone Joint Surg Am. 2013; 95(15): 1420–5. PubMed Abstract \| Publisher Full Text\n\nMcCormick F, Alpaugh K, Nwachukwu BU, et al.: Endoscopic repair of full-thickness abductor tendon tears: surgical technique and outcome at minimum of 1-year follow-up. Arthroscopy. 2013; 29(12): 1941–7. PubMed Abstract \| Publisher Full Text\n\nDomb BG, Botser I, Giordano BD: Outcomes of endoscopic gluteus medius repair with minimum 2-year follow-up. Am J Sports Med. 2013; 41(5): 988–97. PubMed Abstract \| Publisher Full Text\n\nThe Burden Of Musculoskeletal Diseases. Reference Source\n\nAckermann PW: Neuronal regulation of tendon homoeostasis. Int J Exp Pathol. 2013; 94(4): 271–286. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJo CH, Kim JE, Yoon KS, et al.: Platelet-rich plasma stimulates cell proliferation and enhances matrix gene expression and synthesis in tenocytes from human rotator cuff tendons with degenerative tears. Am J Sports Med. 2012; 40(5): 1035–1045. PubMed Abstract \| Publisher Full Text\n\nIshida K, Kuroda R, Miwa M, et al.: The regenerative effects of platelet-rich plasma on meniscal cells in vitro and its in vivo application with biodegradable gelatin hydrogel. Tissue Eng. 2007; 13(5): 1103–12. PubMed Abstract \| Publisher Full Text\n\nZhang J, Wang JH: PRP treatment effects on degenerative tendinopathy - an in vitro model study. Muscles Ligaments Tendons J. 2014; 4(1): 10–7. eCollection 2014. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang JH: Can PRP effectively treat injured tendons? Muscles Ligaments Tendons J. 2014; 4(1): 35–7. eCollection 2014. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDoss A: Neotendon infilling of a full thickness rotator cuff foot print tear following ultrasound guided liquid platelet rich plasma injection and percutaneous tenotomy: favourable outcome up to one year [v1; ref status: indexed, http://f1000r.es/xz]. F1000Res. 2013; 2: 23. eCollection 2013. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "6747", "date": "24 Nov 2014", "name": "Nikolaos Gougoulias", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author presented the outcome of one case of tendon healing, followed for one year, after PRP injection. First I would like to comment on the terminology. \"Tenotomy\" may be a misleading term. Tenontomy means \"cutting a tendon\". Is this what the author did? Or just an injection. This needs clarification. Furthermore, one cannot prove that it was the PRP that aided tendon healing, or the injection itself. Thee are various good quality randomized studies that showed no difference in the outcomes of PRP versus placebo injections. These should be cited in the discussion. A single case is not even indicative of the effectiveness of PRP. In theory PRPs may have anti-inflammatory and reparative properties, may have shown \"promising\" results in the lab, but their clinical effectiveness is questioned and controversial. Taking into consideration that their use is financially beneficial to doctors and the industry, their use cannot be widespread, unless good quality evidence is available.", "responses": [ { "c_id": "1091", "date": "24 Nov 2014", "name": "Arockia Doss", "role": "Author Response", "response": "First of all, thank you for your time with valuable comments and review.Tenotomy can be performed by a percutaneous technique with multiple points of fenestrations through a tendon (Chiavaras MM, Jacobson JA. Ultrasound-guided tendon fenestration. Semin Musculoskelet Radiol. 2013 Feb;17(1):85-90. doi: 10.1055/s-0033-1333942. Epub 2013 Mar 13. Review. PubMed PMID: 23487340), (Housner JA, Jacobson JA, Misko R. Sonographically guided percutaneous needle tenotomy for the treatment of chronic tendinosis. J Ultrasound Med. 2009 Sep;28(9):1187-92. PubMed PMID: 19710216).With a tenotomy the tendon is fenestrated subcutaneously in an attempt to change a chronic tendinopathy to an acute condition, thus allowing the opportunity to heal. This is a distinctly different procedure to an 'injection' where the needle is not moved within the tendon. It is made clear in the paper that this single patient case report does not constitute evidence for routine application. The purpose of this paper is to document tendon tear repair seen on ultrasound in conjunction with clinical improvement. This should form the basis of future rigorous studies on efficacy of a combined technique of PRP tenotomy, PRP alone or tenotomy alone, versus placebo.Such case reports offer practice based evidence rather than evidence based practice." } ] }, { "id": "6745", "date": "16 Dec 2014", "name": "Jason K.F. Wong", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI think this is a controversial area whereby it is not possible to evaluate the effectiveness of PRP specifically on the patients improvement in symptoms. We all have patients who respond in some way to a change in treatment, behaviour or management which do not work again when trialed. There are too many confounding factors here that make this observation uncertain, for example the patient could have got better without treatment, the needling could have caused the site to bleed and the clotting could have healed the defect, fluid injected itself could have caused it to heal, the US scan is not confirmation of neotendon as organised scar can have similar appearances and a biopsy would have to be performed to confirm. Many different possibilities exists that without proper study and experimental design means that this is an interesting observation which may be biased and incorrect. Based on the literature published in this field to date, it cannot be supported to change clinical practice without a proper randomised control study based on significantly higher patient numbers. I know that this practice goes on in many sports clinics at much financial gain to independent practicing sports clinicians.", "responses": [ { "c_id": "1163", "date": "07 Jan 2015", "name": "Arockia Doss", "role": "Author Response", "response": "I wish to thank the reviewer for his time to write a review for this case report on the repair of a gluteal tendon tear following ultrasound image guided Platelet Rich Plasma (PRP) tenotomy. It is regrettable that this reviewer has not approved this case report. Approval and indexation in Pubmed is crucial for furthering study in a new area of controversy and interest. A similar case report on tendon repair in rotator cuff tear published in F1000Research was subsequently approved and indexed in PubMed1. The reviewer states that there is not enough evidence of efficacy data from a single patient related outcome and there is lack of evidence that the treatment actually worked. He also states that PRP procedures are performed for financial gain and his approval of this paper may result in widespread routine clinical use of PRP. I wish to make the following points in response to this review. Purpose of Case Reports Case reports such as this article, serve the purpose of describing novel, innovative or new treatment options by describing the outcome in a single or series of cases. Case reports do not offer ‘data’ on efficacy. Case reports offer qualitative descriptions that reflect day to day clinical practice. There are many variables leading to bias in a case report. Controlling bias and proving efficacy of such a new treatment is the purpose of controlled studies and trials. Case reports are meant to be the subject of future rigorous studies and are not meant to change routine clinical practice. This is made very clear in my article. The weight of establishing efficacy rests on future controlled studies. Until such time, ultrasound guided platelet rich plasma (PRP) tenotomy in gluteal tendon tears should not be used in routine clinical practice without audit and follow up arrangements. This is also made very clear in my paper. Therefore, to ‘Not Approve’ this case report on the basis of ‘lack of efficacy’ suggests that the reviewer has not taken into account the very nature and purpose of case reports and has not provided due credit to the points that were acknowledged in my article anyway. One should not forget that case reports described the first heart transplant for cardiomyopathy 2 and the self experimentation by Barry Marshall with Helicobacter pylori to prove the pathogenesis of peptic ulcers 3. Is there a need for a tendon biopsy ? The reviewer suggests the need for biopsy to prove healing of the treated tendon tear. When patients improve after treatment of a tendon, a biopsy to prove regeneration of a healed tendon is impractical, unnecessary and may be harmful. The British Medical Journal published a case report on tendon regeneration in a case of rotator cuff tendon tear following percutaneous treatment 4. Good clinical outcome coupled with restoration of normal tendon appearances on magnetic resonance imaging formed the basis of the conclusion of ‘tendon regeneration’ by the authors. Regeneration of tendon was concluded without tendon biopsy in that case report. Why is this case report important in gluteal tendon tears? The concept of being able to repair a tendon with a relatively inexpensive out of hospital procedure is innovative. This innovative approach has not been given due credit by the reviewer. Tendon repair is currently perceived as being a surgical procedure in a hospital. Surgical tendon repair procedures are performed in private hospitals for those with private health fund insurance schemes in the Western world. This means that patients without private health cover do not have access to a hospital based surgical procedure and are left to live in pain for a long time. This leads to a sedentary life style and associated co-morbidities in a large proportion of patients. Irrespective of currently available surgical or non surgical options, tendon disorders are a major cause of morbidity in the western world and any innovation should be welcome. There is a move towards keeping patients out of hospital due to escalating costs and risks of infection from antibiotic resistant microorganisms in a hospital environment. This case report is timely and reflects the current trend of out of hospital procedures that may potentially offer an alternative to in hospital surgical repair. Reports such as this offer proof of concept of regenerative percutaneous tendon repair procedures. Peer review BiasOpen access publishing with open peer review offers a platform for unbiased dissemination of articles irrespective of the source of the article - whether the article is from a small private practice or from an academic department. Richard Smith (Former Editor, British Medical Journal) describes the bias of accepting and publishing articles from big name institutions as the Mathew effect: `To those who have, shall be given; to those who have not shall be taken away even the little that they have’ 5. The comment ‘sports physicians performing PRP procedures with much financial gain’ suggests such a bias of the reviewer against private practices. The reviewers’ comment assumes that private practices perform such procedures for the revenue from such procedures. This is a regrettable and largely incorrect view. Such new procedures are offered by private practitioners or demanded by patients due to the lack of other options in an area of unmet medical need, as patients have tried existing options with no benefit. The comment implies that good medical write ups are not to be expected from private non academic practices. Purpose of this review The comment ‘based on the literature published in this field to date, it (PRP) cannot be supported to change clinical practice without a proper randomised control study’ suggests that the reviewer rejects this case report on the basis of other studies and the possibility that PRP may be used widely in clinical practice. I find that such a conclusion does not serve the purpose of this review. ConclusionThe weight of careful responsible reviews rests on all of us who have the ability to change medical practice on the basis of good evidence. Where there is no such evidence in an area of unmet medical need as seen with tendon disorders that result in a huge health economic burden, should we not provide practice based evidence? When such practice based evidence is written up, we need a review without any prejudice to the source of the article. Unfortunately this review does not help us take a forward step to reduce the health burden from musculoskeletal disorders. References 1. Doss A: Neotendon infilling of a full thickness rotator cuff foot print tear following ultrasound guided liquid platelet rich plasma injection and percutaneous tenotomy: favourable outcome up to one year [v1; ref status: indexed, http://f1000r.es/xz]. F1000Researcg. 2013; 2 (23). PubMed Abstract \| Free Full Text \| Publisher Full Text \| Reference Source 2. Barnard CN: The operation. A human cardiac transplant: an interim report of a successful operation performed at Groote Schuur Hospital, Cape Town.S Afr Med J. 1967; 41 (48): 1271-1274 PubMed Abstract3. Marshall BJ, Armstrong JA, McGechie DB, Glancy RJ: Attempt to fulfil Koch's postulates for pyloric Campylobacter. Med J Aust. 1985; 142 (8): 436-439 PubMed Abstract4. Wang AW, Bauer S, Goonatillake M, Breidahl W, et al.: Autologous tenocyte implantation, a novel treatment for partial-thickness rotator cuff tear and tendinopathy in an elite athlete.BMJ Case Rep. 2013. PubMed Abstract \| Publisher Full Text \| Reference Source 5. Smith R: Peer review: a flawed process at the heart of science and journals. J R Soc Med. 2006; 99 (4): 178-182 PubMed Abstract \| Free Full Text \| Reference Source" } ] } ]	1	https://f1000research.com/articles/3-284
https://f1000research.com/articles/3-172/v1	25 Jul 14	{ "type": "Research Article", "title": "Characterization of M-laurdan, a versatile probe to explore order in lipid membranes", "authors": [ "Serge Mazeres", "Etienne Joly", "Andre Lopez", "Catherine Tardin", "Etienne Joly", "Andre Lopez" ], "abstract": "Microdomains corresponding to localized partition of lipids between ordered and less ordered environments are the subject of intensive investigations, because of their putative participation in modulating cellular responses. One popular approach in the field consists in labelling membranes with solvatochromic fluorescent probes such as laurdan and C-laurdan. In this report, we describe a high-yield procedure for the synthesis of laurdan, C-laurdan and two new fluorophores, called MoC-laurdan and M-laurdan, as well as their extensive photophysical characterization. We find that the latter probe, M-laurdan, is particularly suited to discriminate lipid phases independently of the chemical nature of the lipids, as measured by both fluorescence Generalized Polarization (GP) and anisotropy in large unilamellar vesicles made of various lipid compositions. In addition, staining of live cells with M-laurdan shows a good stability over time without any apparent toxicity, as well as a wider distribution in the various cell compartments than the other probes.", "keywords": [ "laurdan", "fluorescent probes", "lipid phases", "Generalized Polarization", "anisotropy" ], "content": "Introduction\n\nNumerous physiological processes take place at the cell plasma membrane and its organization into domains participates in modulating many cellular responses1. In animal cells, plasma membranes (PMs) are mainly composed of phosphatidylcholine, sphingomyelin and cholesterol2. Studies on in vitro model systems such as membranes isolated from cells or liposomes prepared either with synthetic lipids or extracted from PMs of cells from various sources have been instrumental in understanding the formation of lipid domains in biological membranes3,4. For example, pure sphingomyelin is known to form solid like phase (So) at physiological temperature5, due to its long acyl chain. In cell membranes containing cholesterol, however, cholesterol and sphingomyelin have been shown to interact and form liquid ordered phases (Lo) surrounded by a liquid disordered phase (Ld) essentially comprised of phosphatidylcholine2.\n\nResults obtained with artificial model membranes are, however, difficult to transpose to natural membranes and research performed on intact live cells is preferred6. In this context, fluorescent probes incorporated directly into cells are used extensively to reveal and characterize lipid domains, at sufficiently low concentrations that they should cause minimal disturbance to the membrane organization7,8.\n\nFor labelling biological membranes, two main classes of fluorescent molecules are usually used8. The first one is composed of lipids such as phospholipids, sphingolipids, gangliosides or cholesterol that are attached to classical fluorescent dyes like fluorescein isothiocyanate (FITC) and nitro benzoxadiazol (NBD)9,10. Such probes can be used to image domains because of their preferential partitioning to certain phases in models as well as in cell membranes11. The phase preference of these dyes is a subtle equilibrium between the fluorescent part of the molecule, the acyl chains and the interaction with the surrounding lipids of the membrane, thus some narrow changes in molecular interactions may shift the probe partitioning12,13. The second class of dyes developed for studying order in biological membranes are molecules with fluorescent moieties which are highly sensitive to the surrounding micro-environment. One of the most commonly used families of probes is laurdan and its derivatives14,15, whilst other probes include Nile Red16, di-n-ANEPPDHQ17, 3-hydroxy-flavone18 and 2-anthroyl lipid derivatives19. For such probes no preferential phase partitioning is expected.\n\nLaurdan, and other related fluorescent molecules containing 2-hydroxy-6-dodecanoyl naphthalene are environment-sensitive dyes which exhibit a large Stokes shift correlated to the polarity of the surrounding medium20,21. This effect comes from intramolecular charge transfer (ICT) when solvent relaxation occurs, as well as from local excitation (LE), without solvent relaxation22. In polar solvents, the emission is red shifted compared to apolar ones23. When laurdan is embedded in lipid bilayers that contain no sterol, a transition from solid (So) to liquid disorder (Ld) phases results in a large red shift20. The fluorescence modulation can originate from complex processes including reduced solvent relaxation due to lipid packing and high micro-viscosity, abrupt water molecules gradient through lipid bilayers24, possible H-bonding with donor lipid groups25 and/or dyes bending and sliding along the z-axis26. Complex lipid mixtures including sphingomyelin and cholesterol can also modulate the fluorescence parameters due to specific lipid/lipid and lipid/dye interactions27. Recently, Kim et al.15 synthesized and characterized C-laurdan, which also shows great sensitivity to changes in lipid order in the bilayers, but labels the plasma membrane of cells more efficiently than laurdan. Because C-laurdan is not commercially available, we decided to synthesize it ourselves. To achieve this, we revisited the synthesis of 2-hydroxy-6-dodecanoyl naphthalene, the precursor of laurdan, which also provided us with access to two new candidate probes molecules that we called M-laurdan and MoC-laurdan, in addition to laurdan and C-laurdan. Here we report the thorough characterization of the photophysical properties of these four probes in parallel, both in solvents and in model membranes. We also show their possible use to label live tissue culture. We show that, among the four candidate probes, M-laurdan looks as a particularly interesting alternative to laurdan or C-laurdan to label live cells.\n\n\nMaterials and methods\n\nChemicals and solvents were all purchased from Sigma-Aldrich and ultra-pure water was used for buffer preparation (Milli-Q 18 MΩ, Millipore, France).\n\nA series of media covering a range of dielectric constants from 2 to 60, corresponding to the variation of the polarity of a bilayer from the center to the lipid head group, was obtained by mixing the right amount of dioxane in water. The dielectric constant was taken as the linear combination of a mixture of non-polar (1,4-dioxane, ε20°C=2.2) and polar (Water, ε20°C=80.1) solvents according to their molar fraction28. Other experiments were carried out using pure solvents with dielectric constant ranging from 4.8 to 33 and classified as non-polar (chloroform), polar aprotic (dichloromethane, acetone, acetonitrile, dimethylformamide) and polar protic (ethanol, methanol) media. For all experiments, the dyes were dissolved in solvents to a final concentration of 1 µM, starting from dried residues.\n\nDPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and cholesterol were purchased from Sigma-Aldrich. Porcine brain sphingomyelin (BSM, containing 50% C18:0 and 21% C24:1 as major compounds) and egg sphingomyelin (PSM containing 86% C16:0 as major compound) were from Avanti Polar Lipids. Large Unilamellar Vesicles (LUVs) were obtained by sonication. The amounts of chloroform stocks of lipids and dyes, calculated to obtain a final concentration of 100 µM for the lipids and 1 µM for the probes, were mixed in glass tubes. The chloroform solvent was first evaporated under nitrogen flow and then under vacuum for 2 hours. 3 ml of MOPS buffer (3-(N-morpholino) propane sulfonic acid, 10 mM, pH 7.3, NaCl 100 mM, EDTA 10 µM) was then added (at a temperature 5°C above the gel-to-liquid phase transition Tm for DPPC, BSM and PSM, and at room temperature for the other lipids and lipid mixtures). The tubes were then sonicated for 5 minutes (immersion tip diameter 3 mm, delivered power 18 W, Bioblock, France) and kept at 4°C overnight before use. Characterization by dynamic light scattering (DLS) of the lipid vesicles showed the main population of vesicles had sizes ranging from 150 to 350 nm in diameter depending on the mix of lipids used to prepare the liposomes. The OD at 500 nm was checked systematically, to ensure that it remained below 0.05 to avoid any inner filter effect. The compositions of the lipid mixtures were chosen on the basis of the literature (see Table S1).\n\nAbsorption spectra were recorded on a Specord 205 spectrophotometer (Analytik Jena, Germany), fluorescence spectra on a FLSP920, lifetimes on a TCSPC Model 199 (both from Edinburgh Instruments, UK) and anisotropy on an automated home-built set-up allowing to control and change the temperature of the sample.\n\nFluorescence emission spectra were recorded over the range from 370 to 600 nm with excitation set to 360 nm on a thermostatic sample holder. Lifetimes were recorded at 460 nm, with 380 nm pulsed LED for excitation (PLS 370, Picoquant, Germany). POPOP was used as a reference for quantum yield (Φ=0.97 in cyclohexane) and lifetime (τ=1.35 ns in ethanol)29. Anisotropy was recorded by integrating the fluorescence emission through a band pass filter (450/50) and polarizers with appropriate orientations (excitation set to 360 nm) at temperatures ranging from 10 to 60°C (2°C step increments).\n\nQuantum yield was calculated using the following equation29:\n\n\n\nwhere S is the area of the integrated intensity, OD is the optical density and n is the refractive index (r for reference sample).\n\nFluorescence decay times were analyzed as a sum of exponentials using a least-squares algorithm according to the following equation30:\n\n\n\nin which Ii is the steady-state intensity and τi is the lifetime (Iiτi=α1).\n\nMean fluorescence lifetime was calculated as follows31:\n\n\n\nwere is αi the normalized pre-exponential factor and τi is the lifetime of the decay i.\n\nThe reported anisotropies were corrected for instrument response (G-factor) and calculated using the following expression32:\n\n\n\nwere Ixy corresponds to the fluorescence intensity recorded for vertical (v) or horizontal (h) polarizer position (x for excitation path, y for emission path).\n\nThe Generalized Polarization (GP) was calculated from the measured fluorescence intensities at 440 and 490 nm33:\n\n\n\nCOS7 cells, obtained from ATCC, were grown in DMEM + 10% fetal calf serum and passaged 1/10 by trypsinization every three or four days. For microscopy, COS7 cells were left to adhere overnight on sterile glass coverslips (Thermo Scientific Menzel). Cells were then rinsed twice with PBS with no serum, before incubating at 37°C with the probes diluted to 10 μM in PBS with no serum, for 15 minutes for C-laurdan and 30 minutes for the other three. The cells were then rinsed twice with PBS + 10% FCS warmed to 37°C.\n\nCell imaging was performed on a LSM 710 NLO-Meta confocal microscope with spectral detection (Zeiss, Germany) coupled to a two-photon laser source (Chameleon Vision II, Coherent, France). Images were taken through a 40x/1.2W objective under controlled environment (37°C, 5% CO2). Images were recorded in one pass from 420 to 600 nm with 10 nm steps (excitation, 720 nm). GP maps were calculated with ImageJ (NIH) from images recorded at 440 and 490 nm using the formula described above and a custom-built macro (available upon request).\n\n\nResults and discussion\n\nTo simplify and ameliorate the synthesis of the laurdan family dyes, we revisited the synthesis procedure of their precursor, 2-hydroxy-6-dodecanoyl naphthalene, by taking advantage of a Fries rearrangement in methane sulfonic acid, as proposed by Commarieu et al.34. Using commercially available standard reactants, naphthol-2 and lauroyl chloride, this precursor was produced with a yield of nearly 100% in two reaction steps including the Fries rearrangement.\n\nBased on this common precursor, four fluorescent probes related to laurdan were obtained: 6-dodecanoyl-2-(dimethylamino) naphthalene (laurdan), 6-dodecanoyl-2-(methylamino) naphthalene (M-laurdan), 6-dodecanoyl-2-[N-methyl-N-(methoxycarbonyl) amino] naphthalene (MoC-laurdan) and 6-dodecanoyl-2-[N-methyl-N-(carboxy-methyl) amino] naphthalene (C-laurdan) (Figure 1). Laurdan was produced using dimethylamine. M-laurdan, MoC-laurdan and C-laurdan were produced following the protocol reported by Kim et al.15. All dyes were produced with satisfactory yields using conventional chemistry facilities. Finally, the four compounds were purified by thin layer chromatography and characterized by 1H-NMR (see SI attribution peaks). Dyes formulae, molecular weight and basic photophysic characteristics are presented in Table 1.\n\nReagents and conditions: a] Naphthol-2, Lauroyl chloride (triethanolamine); b] Fries rearrangement, Methane sulfonic acid; c] (CH3)2NH HCl, Na2S2O5, NaOH, H2O; c'] CH3NH2 HCl, Na2S2O5, NaOH, H2O; d] BrCH2COOCH3, Na2HPO4, CH3CN; e] KOH, EtOH.\n\nCalculated using C=12.011, H=1.008, N=14.007 and O=15.999 (atomic weights). ε measured at 360 nm in CHCl3. **Φ measured in CHCl3.\n\nIn order to evaluate the possible influence of the nature of the solvent beyond its dielectric constant on the photophysical properties of the four probes, we carried out measurements in dioxane/water mixtures with dielectric constants spanning a broad range of values (Figure S1), and next in a variety of pure solvents with dielectric constants ranging from 4.8 to 33 (Figure 2 and Table 2). From the steady-state measurements in dioxane/water mixtures, for all four dyes, we found a linear correlation between the measured GP and the dielectric constant of the media (Figure S1). In contrast with these results obtained in dioxane/water mixtures, low values of GP were seen only in the pure solvents ethanol and methanol, which are both polar protic solvents with dielectric constants above 20 (εEtOH=25 and εMeOH=33). The other photophysical characteristics, quantum yield and lifetime (Φ, τ), were found to be rather constant for all the probes except for C-laurdan, for which the quantum yield was seen to decrease very significantly in protic solvents and polar aprotic solvents with ε>20.\n\nBlue, non-polar solvent (◆ chloroform), red, polar aprotic solvents (■ dichloromethane, ◆ acetone, ▲ acetonitrile, ● dimethylformamide), green, polar protic solvents (▲ ethanol, ◆ methanol). Experiments were performed twice and reported values are mean ± SD (not visible error bars are below the size of symbols).\n\nε refers to the solvent dielectric constant, quantum yields ϕ are measured using POPOP as reference (ϕPOPOP=0.97 in cyclohexane), lifetimes τ are recorded at 460 nm (excitation, 380 nm) and reported along with the goodness of the fit χ2 (POPOP was used as reference for deconvolution, τPOPOP=1.35 ns in ethanol), kr (ϕ/τ) and knr ((1-ϕ)/τ) are the deexcitation constants deduced from quantum yields and lifetimes, Generalized Polarization, GP, are calculated from emission intensities at 440 and 490 nm.\n\nFor all four probes, the GP was low only in protic solvents, i.e. ethanol and methanol (Figure 2), presumably because of those solvents’ capacity to make hydrogen bonds. The Lippert plots of the four probes were found to be linear with a similar slope, which leads us to conclude that the different dyes are similarly sensitive to solvent polarity (Figure S2).\n\nNext, we characterized the fluorescence of the four dyes in water at a final concentration of 1 µM. For this, we used two procedures of solubilization: either directly from dried dye residues, or indirectly, using dimethyl sulfoxide (DMSO) loading (Figure S3). For the indirect procedure, similarly to what is commonly performed for labelling cells, dye stock solutions were prepared at 1 mM in DMSO and injected at 1:1000 into water7. For laurdan and M-laurdan, no fluorescence and extremely low absorption were detected in the solution obtained directly from dried residues. In the solutions obtained by DMSO loading, very weak fluorescence signals were detected, which may correspond either to slightly improved solubility or to the probes remaining in complexes with DMSO. Our data are in agreement with the well-known insolubility of Laurdan in water35. The fluorescence intensity of the MoC-laurdan obtained from dried residue was significant but with a very marked blue shift, suggesting that dyes were not surrounded by water molecules and quite possibly aggregated. For the MoC-laurdan solution obtained by DMSO loading, the fluorescence was very high, with the emission maximum near 460 nm at the time of the measure. This suggested that the probe remained in an environment with a lower dielectric constant than pure water and thus probably remained conjugated to some DMSO molecules for extended periods of time. For C-laurdan, Kim et al.15 have previously reported that this dye is soluble in water. Accordingly, we detected an intense fluorescence, with the spectra being independent of the solubilization procedure utilized. The emission spectrum was red-shifted, with a maximum at 520 nm compared to 425 nm in chloroform, which could correspond to the dye sensing a protic surrounding.\n\nAltogether, we conclude that i) laurdan and M-laurdan are probably almost insoluble in water when starting from dried residues, and have very low fluorescence in water when injected with DMSO, ii) MoC-laurdan can fluoresce in pure water, but may either aggregate if placed in water directly, or remain associated to DMSO when this is used to dissolve the probe iii) C-laurdan is soluble and fluoresces in pure water.\n\nFor labelling model bilayers, we employed the most adequate procedure consisting of directly mixing lipids and fluorescent probes in chloroform, followed by steps of desiccation and rehydration in buffer that lead to the direct assembly of fluorescently labelled LUVs. In contrast, this direct procedure cannot be applied to label live cell membranes. An indirect procedure is then applied where the fluorescent solvatochromic lipid probes are commonly solubilized into DMSO before large dilution in serum-free culture medium and addition to cells36.\n\nUsing laurdan and C-laurdan on LUVs made of DPPC, DPPC/cholesterol (6:4) and POPC, we performed GP and anisotropy measurements to compare both types of samples and we found no detectable difference, suggesting that the presence of low concentrations of DMSO in the samples does not have any significant effect on this kind of probes (Figure S4).\n\nNext, we examined the capacity of the four probes to label LUVs, using the common procedure of adding them solubilized in DMSO as it is the case for live cell membrane labelling. We first estimated the time required for the insertion of 50% of the fluorophores from the measurement of fluorescence intensity over time after injection of dyes on LUVs made of DPPC/cholesterol (6:4) (Figure 3A-left panel). For laurdan, M-laurdan and MoC-laurdan, fluorescence was found to increase over time, to reach a plateau around 60 minutes, with the first fluorescence measurement recorded 1 minute after injection being close to zero. From our data, we estimated the half insertion times to be in the range of three to 6 minutes for M-laurdan, and 10 to 15 minutes for laurdan and MoC-laurdan. In contrast, C-laurdan fluorescence intensity recorded 1 minute after injection was already high and stayed constant during the course of the experiment. For MoC-laurdan, the maximum value of the emission spectra shifted from 460 to 425 nm over the course of the experiment, suggesting that the probe was still mostly conjugated with DMSO at the early time points (< 5 minutes) and inserted progressively in the bilayer at later times. The large differences observed between the insertions times of the various probes probably originate from differences in their hydrophilic head volumes. All in all, however, the insertion times of all four probes are compatible with membrane labelling of live cells.\n\nA (Left panel): Incorporation of the dyes into LUVs resulted in an increase of the fluorescence intensity over time. Unlabeled LUVs made of DPPC/chol (6:4) (100 µM, sample volume 3 ml) were mixed with dyes dissolved in DMSO. The final dye concentrations were 1 µM and the DMSO/water ratio 1:1000. Measurements were carried out at 20°C and fluorescence intensities were recorded at 440 nm. The fitting curve was obtained using a one site binding equation. B (Right panel): For flow cytometry (FACS), cells were harvested as a single cell suspension in tissue culture medium + serum after trypsinization and washed twice with PBS with no serum. For each probe, 1 µl of stock solution at 2 mM in DMSO was mixed with 2 million cells in 200 µl PBS (10 µM final) at room temperature. After 10 minutes, the tubes were placed in a water bath at 37°C for 30 min, before cells were washed twice in 5 ml and resuspended in a final volume of 1 ml with PBS + 10% serum warmed to 37°C. Cells were then further incubated for 20 min at 37°C. Every 10 minutes throughout the procedure, the volume corresponding to 250.000 cells was taken from each sample, placed on ice in a final volume of 500 µl ice-cold PBS + 10% serum, and submitted to FACS analysis on a LSR II flow cytometer with a UV laser and using the channel with a 450/50 BP filter.\n\nTo record the bilayer characteristics measured with the four probes we chose two phospholipids: POPC, a major component of biological membranes as well as DOPC, a phospholipid seldom encountered in nature, but which is broadly used as a model to generate raft-like domains in membranes37. At room temperature, pure POPC and pure DOPC are known to assemble into Ld phases38,39. Accordingly, GP values, as a function of temperature, remained low for all probes, in agreement with the high level of hydration of the lipid bilayer in Ld states (Figure 4). GP measured on pure DOPC were systematically below those measured in pure POPC, indicating a higher degree of hydration in DOPC bilayers.\n\nWhen anisotropy was measured, as a function of temperature, laurdan and M-laurdan gave similar values, whereas lower values were found for MoC-laurdan and higher for C-laurdan (Figure 4). This could stem from a difference in the transverse positioning of the dye into the bilayer affecting their rotational mobility, or from different fluorescence lifetimes.\n\nThe fluorescence lifetimes were thus measured, and found to be very similar to one another for the first three: laurdan (<τ>POPC=3.19 ns, <τ>DOPC=3.04 ns at 20°C), M-laurdan (<τ>POPC=3.16 ns, <τ>DOPC=3.33 ns at 20°C) and MoC-laurdan (<τ>POPC=3.03 ns, <τ>DOPC=3.17 ns at 20°C). For MoC-laurdan, the lower anisotropy could thus indicate a more superficial insertion of the probe that may have come from its bulky head group. For C-laurdan, fluorescence lifetimes were reduced by 30%, compared to the others (<τ>POPC=2.26 ns, <τ>DOPC=2.13 ns at 20°C) (Table 3), and the higher anisotropy may reflect a more constrained rotational mobility.\n\nIn the table, the lines in blue correspond to bilayers in So phase, in green in Lo, and in red in Ld.\n\nIn addition to these bilayers comprised of single components, we also carried out experiments on a lipid bilayer composed of two components, which has been previously characterized as being in Ld phase at room temperature: DOPC and cholesterol with a 2:1 molar ratio27,40,41. Whilst only few differences were observed on the anisotropy measurements, the GP values were clearly higher than on pure DOPC, in agreement with the well-described capacity of cholesterol to expel water molecules from the bilayer interface42.\n\nWe then turned our interest to the characterization of the four probes in lipid bilayers in solid-like phases (Figure 5). For bilayers made of single components, we chose either pure DPPC, which is classically used for model bilayers, or sphingomyelins, as they are major components of the microdomains observed in cells’ plasma membranes. Two sources of sphingomyelins were used, which carry acyl chains with different lengths: BSM (mostly C18:0) and PSM (mostly C16:0). When incorporated into a DPPC bilayer, all four probes showed a steep decrease for both GP and anisotropy when heated above the melting transition of 41°C, indicating that the four probes are equivalently sensing the phase behavior from So to Ld occurring in the bilayer, in agreement with the results found in the literature for laurdan43. At room temperature, i.e. in a solid DPPC bilayer, the lifetimes measured for the four probes were all much longer than the ones measured in the Ld phase, ranging from 4.86 to 5.82 ns (Table 3).\n\nThe same measurements performed on bilayers made of sphingomyelins gave results which proved more complicated to interpret. First, the fluorescence emission spectra measured in sphingomyelins below the melting transition showed a broader peak when compared to the spectrum obtained with DPPC (Figure S5). As seen in Figure 5, laurdan and M-laurdan exhibited a steep fall in GP around 35°C for BSM44,45 and 41°C for PSM31. By contrast, MoC-laurdan was quite insensitive to temperature transitions in both sphingomyelins, whilst C-laurdan showed a remarkable reduction of amplitude for the PSM transition. For anisotropy measurements, the steep decrease characteristic of the solid to liquid phase transition46 was only seen for laurdan and M-laurdan, whilst MoC-laurdan did not show any significant change of anisotropy induced by the temperature, and C-laurdan only to a reduced extent.\n\nThe strong contrast between the results found with DPPC and sphingomyelins might be related to the differences in their backbone structure since both types of lipids carry phosphocholine as head groups, but DPPC can only accept a single hydrogen bond while sphingomyelin possesses an additional hydrogen bond accepting group. The very weak correlation between anisotropy and temperature observed for MoC-laurdan may be a consequence of the very low values of its fluorescence lifetimes in sphingomyelins (<τ>BSM=2.00 ns, <τ>PSM=2.77 ns at 20°C) compared to DPPC (<τ>DPPC=5.24 ns at 20°C), which may in turn be due to a poor insertion of the dye into the lipid bilayer.\n\nIn sphingomyelin bilayers, M-laurdan, laurdan and to a lesser extent C-laurdan exhibited the expected steep decrease in their GP, with temperature corresponding to a melting transition. The differences observed in the steepness of the curve at the melting transition, as well as in the width of the fluorescence emission spectrum, could also arise from a shallow insertion of the probes in the sphingomyelin membranes due to the repulsion between the amino groups on the sphingomyelins and the one carried by the fluorescent probes, as already proposed for laurdan46. This effect would be reduced for M-laurdan because it has the smallest head group of the family. On the contrary, when the probes were inserted in pure BSM or PSM bilayers, it was only with laurdan and M-laurdan that the fluorescence decay exhibited a lifetime component close to 4 ns at 20°C (Table 3). This longer lifetime, compared to MoC-laurdan and C-laurdan (values less than 3 ns), might explain the better capacity of laurdan and M-laurdan to reveal phase transitions in sphingomyelin bilayers. In fact, the longer lifetimes of M-laurdan and laurdan when those are inserted into bilayers in So phase (between 3.81 and 5.82 ns) may allow anisotropy measurements to provide information about the order parameter of the membrane, since it would be long enough to unravel the frozen state of So phase, as does the diphenylhexatriene DPH with a fluorescence lifetime around 8–10 ns47.\n\nNext, we performed experiments on bilayers formed with DPPC/cholesterol (6:4), a standard mix used to produce lipid bilayers in pure Lo phase48,49. As expected with this model system, no significant differences of GP or anisotropy characteristic of phase transitions was seen with any of the four as of temperature (Figure 6).\n\nWe then prepared the very similar lipid mix as the one used by Kim et al., i.e. DOPC/sphingomyelin/cholesterol (1:1:1), using either BSM or PSM, and confirmed their results showing intermediary values for GP, compared to pure So and Ld phases. Our interpretation, however, differs from theirs. Indeed, we believe that the resulting emission spectrum and the corresponding GP values could simply correspond to a juxtaposition of Ld phases, enriched in DOPC, and Lo phases, enriched in sphingomyelin and cholesterol, (Figure 4 and Figure 6, Figure S5). Similar intermediary values were obtained in POPC/cholesterol (2:1) bilayers, in which Lo and Ld phases are known to coexist. In addition, intermediary values were also found for anisotropy. Furthermore, the interpretation that this mixture, DOPC/sphingomyelin/cholesterol (1:1:1) contains both Lo and Ld phases is in good agreement with previous publications37.\n\nIn line with a hypothesis formulated by one of us50 that solid docks could contribute to the formation of membrane microdomains51, we were interested in measuring whether these probes could discriminate Lo from So phase when both phases were coexisting. For all four probes, coexistence of Lo and So phases in a sphingomyelin/cholesterol bilayer led to the disappearance of the phase transition observed in bilayers made of just sphingomyelin and analyzed using GP. Similar effects were observed by anisotropy. Altogether there thus does not seem to be one particular criterion for any of the four probes which would allow the clear-cut discrimination between So and Lo phases, especially in a biological membrane containing high levels of sphingolipids and cholesterol such as the plasma membrane of eukaryotic cells.\n\nWhen a lipid bilayer goes from fluid to solid, both GP and anisotropy are expected to increase, but those two measurements actually reflect very different characteristics: whilst GP values are mostly influenced by the hydration of the environment, anisotropy reflects on the order parameter of the bilayer related to fluidity. When GP is plotted as a function of anisotropy for measurements made at room temperature on various model membranes (Figure 7), we find the strongest correlation of these two characteristics for M-laurdan, and good correlations for laurdan and MoC-laurdan. For C-laurdan, however, the correlation was only moderate, mostly because of limited variations in anisotropy values between the different environments. In contrast with studies performed with F2N8, another polarity dye27, our data suggest that, when measured with M-laurdan, hydration and fluidity are changing in a correlated manner in all the bilayer compositions tested, including pure sphingomyelins (BSM and PSM) measures falling close to those of DPPC. This result might come from an optimum location of the M-laurdan when inserted into the lipid bilayer as compared to laurdan, MoC-laurdan and C-laurdan, for which the carbonyl function present in sphingosine may interact with the polar heads of the probes, with possible formation of hydrogen bond between the probe and the hydroxyl residue or the amide linkage carried by the sphingosine20. As a result, we think that the simultaneous measurement of GP and anisotropy with M-laurdan used as a probe will permit a true identification of the lipid phases.\n\nExperiments were performed twice and reported values are mean ± SD (unseen error bars are in the size of symbols).\n\nNext, we compared the capacity of all four probes to label live cells. First, we used flow cytometry to determine the speed at which the probes became incorporated into COS7 cells, as well as the speed at which the probes came out of the cells after staining was completed (Figure 3B-Right panel). As expected from what we had found with liposomes, staining with C-laurdan was much faster than with the other three probes, with maximum levels attained after just 20 minutes, while staining with the other probes was still increasing steadily after 40 minutes. The reverse was true, however, when it came to the stability of the staining, with the C-laurdan staining decreasing much more rapidly than for the other three probes. Of note, similar results were obtained when the probes were used to stain either HEK or Jurkat cells, and live gating with propidium iodide did not reveal any particular toxicity on any of the three cell types (unpublished data).\n\nTo perform microscopy, we then used the probes to label COS7 cells adhered to glass coverslips, and labelled them as described in Materials and Methods. The coverslips were then placed at 37°C in the thermostatic chamber of a LSM 710 microscope, equipped for two-photon excitation. Using the spectral capacity of the microscope, the intensity images were recorded in one pass from 420 to 600 nm with 10 nm steps. Images recorded at 440 and 490 nm were used to calculate GP maps (Figure S6). As shown in Figure 8, all four probes resulted in clear labelling of the cells, albeit with remarkably different patterns. With laurdan, the staining was predominantly present in intra-cytoplasmic vesicles. We suspect that those vesicles, which have high GP values, most probably belong to the endosome/lysosome compartment. Of note, a similar pattern of intra-cellular vesicles was also reported by Kim et al.15 on A431 cells. Remarkably, those vesicles were absent from the staining obtained with C-laurdan, which showed a much more diffuse staining pattern, apart from a strong para-nuclear signal of low GP value which probably corresponds to the staining of the endoplasmic reticulum. Both the high GP vesicles and the low GP para-nuclear signal were present in the cells stained with M-laurdan, suggesting that this probe has a more ubiquitous distribution than laurdan or C-laurdan. Staining with MoC-laurdan resulted in patterns somewhat similar to those obtained with M-laurdan, but this probe also gave many undesirable foci on the coverslips outside of cells, which we suspect correspond to precipitated probe aggregates.\n\nUpper row: transmission (Trans.); middle row: total fluorescence (Fluo.) from 420 to 600 nm; bottom row: GP calculated with the values from 10 nm channels centered on 440 and 490 nm (scale bar, 20 µm).\n\nOne feature which is common to all four probes is that the plasma membranes always show higher GP values than the intra-cellular compartments, which is in good agreement with the higher cholesterol content of the plasma membrane.\n\n\nConclusion\n\nWe have developed a simplified approach for the easy and efficient synthesis of 2-hydroxy-6-dodecanoyl naphthalene, the synthesis precursor of laurdan and C-laurdan as well as two new fluorophores, M-laurdan and MoC-laurdan. The measurements of the photophysical parameters performed on those four fluorophores solubilized in solvents showed that, for all of them, hydration has a higher impact than dielectric constant of the solvent. By combining GP and anisotropy measurements on the same model bilayers, we could simultaneously retrieve information related to the hydration level at the interface of the bilayer and to the rotational constraints of the probes. As already reported27, we found that the presence of cholesterol results in reduced hydration of the bilayers, while the presence of sphingomyelin induces both an increase of GP, suggesting an increased hydration in the probes’ environment or extra H-bonding, and an increase of their apparent rotational mobility. This feature is particularly important given that sphingolipids comprise roughly 30% of the lipids in the plasma membranes of eukaryotes52, and they play a critical role in the formation of lipid domains. In our data, M-laurdan, gave results which were best in agreement with the phases expected from the literature independently of the chemical nature of the lipids and of the fluorescence techniques in use. When used to label live cells, M-laurdan was also the probe which gave the most ubiquitous staining patterns, together with a good stability of the staining over time. M-laurdan thus appears as a promising tool for exploring lipid phases and order in biological membranes.\n\n\nData availability\n\nfigshare: Data of M-laurdan characterization to explore order in lipid membranes, doi: http://dx.doi.org/10.6084/m9.figshare.110990153", "appendix": "Author contributions\n\n\n\nSM designed and performed the photophysics experiments and contributed to writing the paper. EJ designed and performed the cellular experiments and contributed to writing the paper. AL designed and performed the chemistry experiments. CT designed experiments and contributed to writing the paper.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nSpecial thanks to Diana Ciuculescu-Pradines (LCC Toulouse, 205 route de Narbonne) for her help providing secure conditions for sensitive chemical reactions, and to Laurence Salomé for her guidance and useful comments. The authors thank the core IPBS facilities, NMR and fluorescence spectroscopy TRI (“Toulouse Réseau Imagerie”, Toulouse, France).\n\n\nSupplementary material\n\n\n\n\nReferences\n\nLingwood D, Simons K: Lipid rafts as a membrane-organizing principle. (New York N.Y). Science. 2010; 327(5961): 46–50. PubMed Abstract \| Publisher Full Text\n\nSimons K, Sampaio JL: Membrane organization and lipid rafts. Cold Spring Harb Perspect Biol. 2011; 3(10): a004697. 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Data Source" }	[ { "id": "5595", "date": "26 Aug 2014", "name": "Vadim Cherezov", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article describes an improved procedure for the synthesis of two fluorescent probes commonly used to image lipid rafts: laurdan and C-laurdan, as well as two new probes: M-laurdan and MoC-laurdan. All four probes are extensively characterized in different solvents and membranes with different lipid compositions in the temperature range from 10 to 60 °C. Based on presented data, the authors conclude that the new probe, M-laurdan, is the best of four for discrimination of lipid membrane phases independently of the chemical nature of lipids. This conclusion appears to stem mainly from the correlation plots between GP and anisotropy shown in Fig. 7. The authors describe correlations as the strongest for M-laurdan, good for laurdan and MoC-laurdan, and moderate for C-laurdan. Just from looking at Fig. 7, however, it is not obvious that the correlation is the strongest for M-laurdan. It would help if the authors will provide a quantitative measure of correlation, such as a correlation coefficient or another similar parameter.The authors also state that M-laurdan makes the best staining patterns in live cells. Based on Figure 8, the difference between laurdan, M-laurdan and MoC-laurdan is not very obvious. It would be great if either a better example or better explanations are provided.Other comments and suggestions are listed below:Some measurements were taken two times and the mean values and standard deviations were provided, e.g. Fig. 2, Fig. 7. Many other measurements seem to be only performed once, for example, Figures 3, 4, 6, S1, S2, S4, Tables 2, 3). I would suggest performing at least 3-4 measurements and report standard deviations for all measured values. Results and Discussion - Insertion of the fluorophores into LUVs: It is stated that no detectable difference is found comparing two methods of labeling referring to Figure 4. However, Figure 4 only shows data for the DMSO loading method and not for direct mixing. Also, it would be great to see the same data for M-laurdan and MoC-laurdan. The section title “Probing model bilayers in pure So states” is misleading, as this section describes mostly transition from So to Ld phase. The same applies to Fig.5, where it says that blue symbols depict So phase. Results and Discussion - Probing model bilayers in Lo states: In the second paragraph Kim et al. should have a reference associated with it.", "responses": [] }, { "id": "5962", "date": "02 Sep 2014", "name": "Guy Duportail", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis work from Mazeres et al. deals with a family of membrane fluorescent probes of the well-known Laurdan family, more precisely Laurdan itself (as “mother” probe), C-Laurdan which was already introduced and studied by Kim et al. (Chembiochem, 2007, 8, 553) and two new derivatives, so-called M-Laurdan and MoC-Laurdan. All these derivatives were synthesized by the authors through simplified chemical steps. The paper mainly presents an extensive characterization of the fluorescence properties of the probes in organic solvents of different polarities and dielectric constants, and in lipid vesicles of varying compositions, focusing on the fluorescence response versus lipid phases, either solid, liquid ordered (raft) or liquid disordered phases. Some preliminary results are also presented concerning live cells labelling. Visualization of lipid domains still remains a challenge, especially in cellular membranes. It is clear that the search and studies of new environment-sensitive membrane probes constitute an important issue in the development of raft imaging tools for cellular applications. In this context, the present work is an interesting contribution to progress in this challenge. However, either to improve the present contribution or to get more clear and precise information in a following work, we have to underline some critical points: Materials and Methods: Large Unilamellar Vesicles (LUVs) were prepared by sonication, as claimed by the authors. Such a method is probably not giving LUVs, but rather, depending on the delivered power, either Small Unilamellar Vesicles (SUVs), or an homogenized distribution of Multilamellar vesicles (MLVs) or Oligolamellar vesicles (OLVs). According to the sizes here presented (150 to 350 nm), I guess we are in presence of the latter case. This is not so important in the present work, since the fluorescence parameters presently collected are responding more or less identically whatever the type of lipid vesicles, but for example if it will be question to study the precise localization of a probe within a lipid bilayer, LUVs must be used. Generally the best procedure to get them is either extrusion or Kachel’s method (BBA,1998,1374, 63). How to interpret fluorescence anisotropy in terms of order parameter? The authors are fully aware that the fluorescence anisotropy ( r ) as a measure of membrane order must take into account the fluorescence lifetime ( τ ) of the probe in the medium. They present a lot of data for both parameters, but in different tables, and the corresponding discussion is hard to unravel. Why not to present the rotational relaxation time parameter ρ which integrates r and τ and is directly correlated with the order parameter through the Perrin-Weber equation [(r0/r) – 1] = 3 τ/ρ (cf. G. Weber, Adv.Prot. Res. 1953, 8, 415). We are presently in an ideal case to use this average parameter, since we are comparing probes presenting the same fluorophore, and consequently more or less the same molecular volume. For further works with these probes, it should be very interesting to proceed to parallax fluorescence quenching experiments in order to know the precise location of these probes (depth of the fluorophore in the lipid bilayer). This would be of great help for the interpretation of several data. In this case, experiments must be performed by using identical and well characterized LUVs. Minor comments and suggestions: Introduction, page 4. It is not true that plasma membranes are mainly composed of PC, SM and Chol. It is true only for the external leaflet. The inner leaflet contains significant amount of PS and to a lesser extent PE. Fig.1: data are obtained in chloroform. Better to indicate in the title than twice in footnotes. Labelling of LUVs, page 6. I do not catch what are “steps of dessication and rehydration”? Is it steps of freezing and defreezing? As compared to the three other probes, C-Laurdan is at least partially ionized, due to its carboxylic group. This explains its water solubility, why its incorporation in lipid vesicles or cell membranes is very rapid, and probably also some of its peculiar data in protic solvents. This should be mentioned. To compare data obtained with DPPC and SMs (either BSM or PSM), we should not forget that SMs are not single molecular species as DPPC, so that it is not surprising that, for example, a phase transition followed by fluorescence anisotropy cannot be so clear-cut with SMs as with DPPC. In their conclusion, the authors are presenting the ubiquitous staining patterns of M-Laurdan in cells as an advantage. It is not so evident as very often a need exists for a selective labelling (for example plasma membrane, or mitochondrial membrane or endosomal membrane).", "responses": [] }, { "id": "5878", "date": "19 Sep 2014", "name": "John D Bell", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper describes the synthesis and characterization of two new forms of the well-known membrane fluorescent probe, Laurdan. The paper is interesting and generally well written. The work is of interest to researchers in the field.I have read the comments of the other reviewers of the paper and agree with their concerns, adding my endorsement of the need to respond to their points. In particular, I also think that Fig. 7 is not a good choice for arguing the superiority of M-Laurdan. As pointed out by Dr. Cherezov, one cannot judge the strength of the correlations without quantitative information. My guess, looking at the figure, though, is that the differences are small. It would, though, be better to compare GP to the rotational relaxation time as suggested by Dr. Duportail since GP and anisotropy are mutually convoluted with excited-state life time.More importantly, it is not clear to me why a strong correlation between the two parameters (GP and anisotropy) is necessarily ideal. Since the two represent different things (polarity of local environment vs. probe wobble (after correcting for lifetime)), isn't there value in exploring conditions under which they may report disparate results? Frankly, I am more excited about the richness of information that could come from comparisons among probes that interact differently with membrane lipids (as revealed by Fig. 5) than by choosing a single probe based on the concept that two different measurements with the probe are best if they are redundant. I would therefore recommend that the paper be steered more in the direction of pointing out the types of information each probe can provide rather than trying to identify one as the optimum.", "responses": [] } ]	1	https://f1000research.com/articles/3-172
https://f1000research.com/articles/3-252/v1	27 Oct 14	{ "type": "Research Article", "title": "Diversity of two-component systems: insights into the signal transduction mechanism by the Staphylococcus aureus two-component system GraSR", "authors": [ "Uzma Muzamal", "Daniel Gomez", "Fenika Kapadia", "Dasantila Golemi-Kotra", "Uzma Muzamal", "Daniel Gomez", "Fenika Kapadia" ], "abstract": "The response to cationic antimicrobial peptides (CAMPs) in Staphylococcus aureus relies on a two-component system (TCS), GraSR, an auxiliary protein GraX and an ATP-binding cassette (ABC) transporter, VraF/G. To understand the signal transduction mechanism by GraSR, we investigated the kinase activity of the cytoplasmic domain of histidine kinase GraS and the interaction with its cognate response regulator GraR. We also investigated interactions among the auxiliary protein GraX, GraS/R and the ATPase protein of the ABC transporter, VraF. We found that GraS lacks autophosphorylation activity, unlike a similar histidine kinase, BceS, of Bacillus subtilis. In addition, the interaction between GraS and GraR is very weak in comparison to the stronger interaction observed between BceS and its conjugated response regulator, BceR, suggesting that CAMP signaling may not flow directly from GraS to GraR. We found that the auxiliary protein GraX interacts with VraF and GraR, and requires the histidine phosphotransfer and dimerization domain of GraS to interact with this protein. Further, VraF requires the GraS region that connects the membrane-bound domain with the cytoplasmic domain of this protein for interaction with GraS. The interactions of GraX with GraS/R and VraF indicate that GraX may serve as a scaffold to bring these proteins in close proximity to GraS, plausibly to facilitate activation of GraS to ultimately transduce the signal to GraR.", "keywords": [ "Two-component systems", "histidine kinases", "response regulators", "GraSR", "Staphylococcus aureus", "cationic antimicrobial peptides" ], "content": "Introduction\n\nStaphylococcus aureus, a Gram-positive coccus, is both a commensal and a major human pathogen. As a commensal organism, it colonizes the skin and nares, and as a pathogen, it causes a variety of infections ranging from superficial skin abscesses to more serious diseases such as pneumonia, meningitis, endocarditis, septicemia, and toxic shock syndrome1. The success of S. aureus as a pathogen relies on its ability to adapt to a wide variety of environmental conditions and to resist host innate immune defense mechanisms2. The extensive use of antibiotics and the adaptability of S. aureus have led to the emergence of multidrug resistant strains in hospital and community settings3.\n\nIn prokaryotes, environmental cues are channeled inside the cell via two-component systems (TCS). A typical TCS is composed of a membrane-bound histidine kinase (HK) sensor and a cognate response regulator (RR) protein. Each organism has a number of these systems that are specialized to respond to a specific cue, despite the conserved nature of domain organization and structural similarities among them4.\n\nThe glycopeptide resistance-associated TCS GraSR, in which GraS is the histidine kinase and GraR is the response regulator protein, regulates the resistance to cationic antimicrobial peptides (CAMPs) in S. aureus5,6. S. aureus resistance to CAMPs involves an increase in the positive cell surface charge through D-alanylation of wall teichoic acid (WTA) and lysinylation of phosphatidyl-glycerol within the cell membrane7,8. Both processes are mediated by enzymes encoded by the dltABCD and mprF operons, respectively. GraR is directly involved in regulation of these two operons6,9,10. Induction of these two operons is selective, and CAMPs such as RP-1 (platelets) and polymyxin B are capable of inducing mprF and dltABCD, but cationic molecules such as vancomycin, gentamicin or calcium-daptomycin are not11.\n\nIn vivo studies showed that sensing and signaling of CAMPs in S. aureus relies on an ATP-binding cassette (ABC) transporter, encoded by the vraFG operon6 and the third gene of graRS operon, graX12. The vraFG operon is regulated directly by GraR, whereas the graXRS promoter is not regulated by GraR. The ABC transporter VraFG is composed of a membrane spanning domain protein, VraG, and an ATP-binding protein, VraF. It is proposed that the ABC transporter senses the presence of CAMPs and transduces the signal through GraSR with assistance from GraX12. Resistance to CAMPs relies on GraR, but not on the ABC transporter, as overexpression of GraR reverses the effect of vraFG, graS, graR or graX deletion. Furthermore, the independence of CAMPs resistance from the ABC transporter suggests that VraG does not function as a detoxification element12.\n\nWhile the ABC transporter is considered to be the sensor for CAMPs, the mechanism of signaling through GraSR and the role of GraS in this process remains unknown. In a typical TCS, the HK becomes active upon sensing an extracellular stimulus in a process that requires phosphorylation of a conserved histidine residue in the cytoplasmic portion of the HK. The information is then transduced to the cognate RR in a second phosphorylation process, whereby a conserved aspartate residue of the receiver domain of the RR becomes phosphorylated. This phosphorylation step modulates the activity of RRs, which often have transcriptional regulatory activities4,13.\n\nHerein, we investigated the autophosphorylation activity of GraS and its interaction with GraR and took a close-up look at the interactions among GraS, GraR, GraX and VraF. For the latter, we cloned, expressed and purified for the first time the GraX and VraF proteins. As a reference in our study of signal transduction mechanism by GraSR, we used a homolog of GraSR in Bacillus subtilis, the BceSR TCS14. There is a 36% sequence identity between GraS/BceS (Figure S1) and 56% sequence identity between GraR/BceR. BceSR is involved in signaling and resistance to bacitracin. Like GraSR, BceSR relies on an ABC transporter for sensing bacitracin; however, unlike GraSR, its ABC transporter also acts as a detoxification element. Both RRs do not regulate the expression of their respective operons14,15.\n\nOur study shows that the cytoplasmic domain of GraS, unlike BceS, does not have autokinase activity and does not interact with GraR. We show that the auxiliary protein GraX interacts with GraR and VraF. In addition, GraX and VraF interact with specific regions of GraS, and we propose that VraF may activate GraS. We see GraX as a bridge between GraS and GraR. Further, we show that there is no cross-talk between GraSR and BceSR, suggesting that, despite the similarities in primary sequences and domain organization between the respective HKs and RRs, other elements may determine the ultimate mechanism of signal transduction in a TCS.\n\n\nMaterials and methods\n\nChemical reagents and materials. Chemicals and antibiotics were purchased from Sigma (Oakville, Canada) or Thermo-Fisher (Whitby, Canada), unless otherwise stated. Chromatography media and columns were purchased from GE Healthcare (Quebec, Canada). Growth media were purchased from Fisher. Escherichia coli strains, NovaBlue and BL21(DE3), and cloning and expression plasmids were purchased from EMD4 Biosciences (New Jersey, USA). The pGEX-4T vector was purchased from GE Healthcare (Quebec, Canada). Restriction enzymes were obtained from New England Biolabs Canada (Pickering, Canada) or Thermo-Fisher. The [γ-32P] ATP was purchased from Perkin Elmer LAS Canada Inc. (Toronto, Canada) or GE Healthcare. The Proteo Extract All-in-One Trypsin Digestion Kit was purchased from EMD4 Bioscience. The genomes of S. aureus strain Mu50 and Bacillus subtilis strain 168 were obtained from Cedarlane (Burlington, Canada). Oligonucleotides were acquired from Sigma (Canada).\n\nThe bceR gene was amplified from B. subtilis strain 168 genome, and graR was amplified from S. aureus strain Mu50 genome. The primer sets used for amplification of each gene are specified in Table 1. Cloning protocols for bceR, graR and graRC were the same. Briefly, each amplicon was ligated to the blunt end sites of pSTBlue-1, and each construct was amplified in NovaBlue cells. The respective plasmids were digested with the appropriate set of restriction enzymes (Table 1), and the inserts were ligated into pET26b(+) at the respective restriction sites. Cloning was confirmed by DNA sequencing (Core Facility, Biology, York University). The pET26b::bceR(or graR, graRC) construct was used to transform BL21(DE3). To clone graRN, we introduced a stop codon after the 134th residue using the Quick-Change mutagenesis kit (Agilent, Mississauga, Canada).\n\nItalicized sequences indicate the restriction sites. Abbreviations: Fwd, forward primer; RE, restriction enzyme; Rev, reverse primer\n\nTo express and isolate the target proteins, cell cultures were grown to exponential phase with an optical density at 600 nm (OD600) of ~0.6, induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, Rose Scientific, Edmonton, Canada), and shaken overnight at 18°C. Cells were harvested at 7,459 × g, resuspended in buffer I (20 mM Tris supplemented with 5 mM MgCl2, pH 7.0 for BceR and pH 7.5 for GraR), and sonicated to liberate the protein. Cellular debris was removed by centrifugation at 18,138 × g for 60 min. The supernatant was loaded onto a DEAE-SepharoseTM column equilibrated in buffer I, and the protein was eluted with a linear gradient of 500 mM Tris supplemented with 5 mM MgCl2 (pH 7.0 for BceR and pH 7.5 for GraR). In the case of BceR, fractions containing the protein were pooled and concentrated using Amicon ultracentrifugation membrane (ultracel 10K, Thermo-Fisher) and then loaded onto a heparin-sepharose affinity column equilibrated with buffer I. Protein was eluted with a linear gradient of 500 mM Tris supplemented with 5 mM MgCl2. For GraR, GraRN and GraRC, as a second step of purification, we employed size-exclusion chromatography. Protein samples were loaded onto Sephacryl S-200 HiPrep 26–60 size-exclusion column (GE Healthcare) equilibrated with 50 mM Tris buffer (pH 7.4) and supplemented with 100 mM KCl and 5 mM MgCl2. The column was run at 1 mL/min. Fractions containing pure protein, as assessed by Coomassie-stained SDS-PAGE, were collected together and concentrated.\n\nProtein concentrations were determined by the Bradford assay. The molecular masses of the isolated proteins were confirmed by electron-spray ionization mass spectrometry (ESI-MS) at the Advanced Protein Technology Centre, Hospital for Sick Kids (Toronto, Canada).\n\nThe nucleic acid sequence encoding the cytoplasmic region of bceS, spanning residues 62 to 334, was amplified from B. subtilis 168 genome. The nucleic acid sequence encoding the cytoplasmic domain of graS, spanning residues 77–346, was amplified from S. aureus Mu50 genome. The same genome was used to amplify the nucleic acid sequence encoding the ATP-binding domain of GraS (GraSCA), spanning residues 181–346. We also amplified the nucleic acid sequence encoding the dimerization and histidine phosphorylation (DHp) domain and the CA domain of graS, spanning residues 110–346. This construct is referred to as GraSDHp-CA. The primer sets used for amplification of the above graS regions are specified in Table 1. We used the pGEX-4T-1 vector to clone the N-terminal GST-fusion proteins of GraS, GraSCA, GraSDHp-CA and BceS. We also cloned the GraS cytoplasmic domain with a hexa-histidine tag on its NH2-terminus, using the pET151/D-TOPO vector.\n\nEach gene was ligated to the blunt-end sites of pSTBlue-1, and this construct was amplified in E. coli NovaBlue. Each plasmid was isolated using the GeneJetTM plasmid extraction kit and digested with the appropriate set of restriction enzymes to liberate the respective insert, which was then ligated into pGEX-4T-1 at the respective restriction sites (Table 1). The pGEX-4T-1::bceS(graS, graSCA or graSDHp-CA) plasmid was introduced into E. coli BL21(DE3). Expression and purification of GST-GraS, GST-GraSCA, GraSDHp-CA or GST-BceS was carried out in the same way. Briefly, protein expression was initiated with 0.5 mM IPTG once the cell cultures reached OD600 ~0.6. Induction proceeded overnight at 18°C. Cells were spun down at 7,459 × g for 20 min, resuspended in 1 × phosphate-buffered saline buffer (PBS, pH 7.4), and then sonicated to liberate the cell contents. Cellular debris was removed by centrifugation at 18,138 × g for 60 min. Purification of each protein was carried out using glutathione-sepharose affinity resin (GE Healthcare). The target protein was eluted with 10 mM reduced glutathione in 50 mM Tris (pH 8.0). Fractions containing the protein were collected together.\n\nExpression of His-GraS was performed in the same manner as for GST-GraS. The cell pellet was resuspended in 20 mM sodium phosphate pH 8.0 buffer supplemented with 300 mM NaCl and 20 mM imidazole. The cells were processed as described above. The supernatant was loaded onto a Ni-NTA (nickel-nitrilotriacetic acid) column (Qiagen), and protein was purified using a linear gradient of imidazole.\n\nThe full length graX gene was amplified from S. aureus Mu50 genome using the primers provided in Table 1. The graX gene was cloned into pET26b between NdeI and HindIII restriction sites. The graX gene was also cloned to the C-terminus of MAT-Tag (HNHRHKH) and -FLAG (DYKDDDDK) epitopes using the pT7 MAT-tagFLAG-1 vector (Sigma). In this case, graX was inserted between the HindIII and XhoI restriction sites of the vector (Table 1). Each expression vector, pET26b::graX or pT7MAT-tagFLAG-1::graX, was introduced into E. coli BL21(DE3).\n\nGraX and MAT-FLAG-GraX were expressed in the same way. Cell cultures were grown to an OD600 ~0.6 at 37°C. At this point, the cultures were cooled at 4°C, induced with 0.5 mM IPTG, and allowed to express protein over 16 hours at 18°C. Cells were harvested by centrifugation at 7,459 × g for 20 min and resuspended in 50 mM sodium phosphate pH 7.2. For MAT-FLAG-GraX, the buffer was supplemented with 300 mM NaCl, and the pH was adjusted to 8.0. Cellular content was liberated through sonication, and the resulting cell lysate was centrifuged at 18,138 × g for 1 hour to remove the cellular debris. For GraX, the supernatant was loaded onto SP-Sepharose cation exchange column. GraX protein was eluted using a linear gradient of 0 to 1 M sodium chloride in 50 mM sodium phosphate pH 7.2. The fractions containing GraX were pooled and concentrated. The protein sample was dialyzed against 50 mM Tris, pH 7.4, 5 mM MgCl2 and 300 mM NaCl. MAT-FLAG-GraX was purified using a Ni-NTA column (Qiagen). The protein was eluted with a linear gradient of imidazole from 10 mM to 300 mM imidazole over 5 column volume.\n\nThe full length vraF DNA sequence (762 bp) was amplified from S. aureus Mu50 genome. The primers designed to clone vraF into the pET24d expression vector harbor the restriction sites of NcoI and BamHI (Table 1). The amplicon was digested with NcoI and BamHI and ligated into the pre-treated pET24d vector with the same restriction enzymes. The resulting construct, pET24d::vraF was introduced into E. coli Nova Blue by heat shock at 42°C. The vraF insertion into the pET vector and the correctness of the gene sequence was confirmed by DNA sequencing (The Centre for Applied Genomics, The Hospital for Sick Kids, Toronto, Canada). The pET24d::vraF construct was introduced into the expression host E. coli BL21(DE3) by heat shock.\n\nTo enable cloning of VraF fused to a 6 × histidine tag (His6-tag) on its C-terminus (His-VraF), we removed the stop codon on vraF to enable translation of a linker region and the 6 histidine tag in pET24d, downstream of vraF. The mutagenesis primers are provided in Table 1. The QuickChange® mutagenesis kit was used to carry out the mutation (Agilent Technology). The mutation was confirmed by DNA sequencing.\n\nUntagged vraF expression was attempted with 0.5 mM or 1 mM IPTG concentration at 18°C or 25°C for 18 hrs, in the absence or presence of 0.5 M sorbitol and 3 mM betaine. However, all these conditions resulted in expression of the protein trapped in inclusion bodies.\n\nOver-expression of the C-terminal His6-tagged vraF was carried out with 0.1 mM IPTG, over 16 hrs by shaking at 16°C. Briefly, 1 mL of overnight grown cell culture of E. coli BL21(DE3) was used to inoculate 1 L of Terrific Broth medium, supplemented with 2.5 M sorbitol and 3 mM betaine. When cell culture reached an OD600 ~0.6, the cell culture was cooled down at 4°C and IPTG was added to a final concentration of 0.1 mM to induce protein expression. The cells were induced over 16 hours at 16°C and subjected to continuous shaking at 200 rpm. They were then collected by centrifugation at 7,459 × g for 20 min. The cell pellet was resuspended in Buffer A (50 mM sodium phosphate, 100 mM NaCl, 10 mM imidazole, pH 7.0 buffer). The cells were lysed by sonication and cell debris was removed by centrifugation at 18,138 × g at 4°C for 1 hr.\n\nPurification of His-VraF was carried out using the batch method. The cell lysate was incubated with 1 mL Ni-NTA resin (Qiagen) for 1 hr at 4°C. The cell lysate-resin mixture was loaded into a column. The resin was washed with Buffer A until no protein was washed out from the column. Elution of VraF was carried out using a step gradient of imidazole in Buffer A: 4 × 1.5 ml 100 mM imidazole, 4 × 1.5 ml 150 mM imidazole; 4 × 1.5 ml 200 mM imidazole and 4 × 1.5 ml 300 mM imidazole.\n\nAutophosphorylation of GST-GraS or GST-BceS was performed as described previously with minor modifications16. Purified GST-BceS, GST-GraS or His-GraS (5 µM) was equilibrated in the phosphorylation buffer (PB: 50 mM Tris, 50 mM KCl, 5 mM MgCl2, pH 7.4) at a final volume of 10 µL. The PB was supplemented with 20 mM CaCl2 (BceS) or 10 mM CaCl2 (GraS). The reaction was initiated by adding [γ-32P] ATP (10 Ci/mmol or 3000 Ci/mmol) at room temperature. Aliquots were removed at different time intervals, and the reactions were quenched by the addition of 5 × SDS sample buffer (2.5% SDS, 25% glycerol, 125 mM Tris-HCL, pH 6.8, 0.0025% bromophenol blue). Samples were analyzed by 12.5% SDS-PAGE. The gels were dried and exposed to an autoradiography cassette, which was scanned using TYPHOON Trio+ (GE Healthcare). The band intensities were analyzed by NIH ImageJ software (version 1.45s). The band intensities were plotted against time, and these curves were referred to as progress curves. The rate constant was determined by plotting the intensity values against a first-order integrated rate law with the equation I = A (1 – e–kt) where, I is the intensity of the band, k is the rate constant, t is time, and A is the proportionality constant between the intensity and concentration of GST-BceS-P. Erithacus GraFit software (version 5.0.10) was used to fit the experimental data. The phosphotransfer between the HK and its cognate RR, and phosphorylation of RRs by small molecule phosphate donors such as acetyl phosphate were carried out as described previously16.\n\nTo investigate whether the purified proteins were folded properly, we collected the CD spectrum (200 – 240 nm) of each protein on a Jasco J-810 instrument (Jasco, Tokyo, Japan) at 22°C using a cuvette with a 0.1cm path length. The CD spectra were collected in 30 mM Tris (pH 7.0) supplemented with 5 mM MgCl2. The final spectra were corrected for buffer contribution.\n\nGlutathione-sepharose affinity resin (75 µL) was equilibrated in 1 × PBS buffer. GST-BceS, GST-GraS, GST-GraSCA or GST-GraSDHp-CA was incubated with the resin for 30 min at room temperature. The flow-through was collected, and the resin was washed with 1 × PBS buffer until no protein eluted. At this point, GST-BceS-bound resin was incubated with BceR at a 1:1.6 ratio, the GST-GraS-bound resin was incubated with GraR at a 1:2 or 1:5 ratio, GraX at a 1:1.3 ratio, or His-VraF at 1:1.3 ratio, the GST-GraSCA-bound resin was incubated with His-GraX at 1:1.6 ratio, or His-VraF at 1:2 ratio, and GST-GraSDHp-CA was incubated with His-VraF at 1:2 ratio. The incubation time in all the cases was 30 min at room temperature. Subsequently, the resins were washed five to seven times with 200 µL of 1 × PBS buffer. The proteins were eluted with 200-µL aliquots of 10 mM reduced glutathione in 50 mM Tris (pH 8.0). Flow-through fractions, wash fractions, and elution fractions were analyzed by 12.5% SDS-PAGE. The immobilized GST-BceS or GST-GraS were also incubated with bovine serum albumin (BSA) to investigate potential non-specific interactions with BceR and GraR, respectively. In addition, the resin itself was incubated with the prey proteins BceR, GraR or GraX to investigate for non-specific interactions of these proteins with resin.\n\nA similar protocol was used to investigate the interaction between GraX and GraR, and His-VraF and GraX. In the former case, MAT-FLAG-GraX was immobilized onto Ni-NTA agarose resin, GraR was added at a 1.2:1 ratio. In the latter case, His-VraF was immobilized onto Ni-NTA agarose resin and untagged GraX was added at 1:2 ratio. The elution fractions were analyzed by 15% SDS-PAGE.\n\nThe oligomerization states of GraR, GraRN and GraRC were determined by the high-performance liquid chromatography (HPLC) size-exclusion column TSK Gel (7.8mm × 30cm, 5µm). The column was calibrated with the standard proteins: aprotonin (6.5 kD), carbonic anhydrase (29 kD), ribonuclease A (37 kD), ovalbumin (45 kD), and conalbumin (75 kD). The molecular masses of the target proteins were determined from the standard curve (log of molecular mass versus retention time). The oligomerization state of GraX was investigated by SDS-PAGE in the presence and absence or dithiothreitol (DTT). The low solubility level of GraX prevented investigation of oligomerization by size exclusion chromatography.\n\nThe promoter region of vraFG (PvraFG), spanning between +28 to -168 with respect to the transcription start site15, was amplified and used to probe the DNA-binding activity of GraR. The graSR promoter region spanning -115 to +75 was amplified using the primers Dir-5′-CGGAATTCATTGAAATGAAATTTTCTACA TC-3′ and Rev-5′-CGGGATCCTTTAGGTTTCATCTAAAATACTCC-3′. Prior to amplification, the primers were 5′ end-labeled with [γ-32P]ATP (3000 Ci/mmol) using T4 polynucleotide kinase. The DNase I footprinting was carried out as described previously17. The gels were dried and exposed to an autoradiography cassette, which was scanned using TYPHOON Trio+ (GE Healthcare). The footprinting gels were analyzed by NIH ImageJ software (version 1.45s). The DNase I footprinting data were used to assess the dissociation constant as the GraR concentration that provided 50% protection.\n\n\nResults\n\nGraS (346 amino acids) and BceS (334 amino acids) are similar HKs; they share 36% sequence identity and both use an ABC transporter for signaling14,15. They consist of a membrane bound domain (BceS: spanning residues 1–55; GraS: spanning residues 1–63) and a cytoplasmic domain referred to as the kinase domain (BceS: spanning residues 105–336; GraS: spanning residues 110–346). We used the amino acid sequence alignments of both proteins to determine the N-termini of each protein construct so that similar regions of these proteins were cloned. Cloning of similar regions of the cytoplasmic portions of GraS and BceS will allow a direct comparison of their functions. The cytoplasmic domains of GraS (77–346) and BceS (62–334) were independently fused at the COOH-terminus of GST, and the proteins were purified to homogeneity. Two other constructs of GraS were fused to the COOH-terminus of GST: GraSDHp-CA, spans residues 110 to 346 and lacks the membrane domain and the linker region; and GraSCA, spans residues 181 to 346 and harbors only the ATP-binding domain of GraS. In addition, the cytoplasmic domain of GraS was also fused to a hexa-histidine tag at its NH2-terminus.\n\nCloning of bceR, graR, graRN, and graRC encoded, respectively, proteins with no extra amino acids on their NH2- or COOH-termini. The proteins were purified in two steps. GraR, GraRN, and GraRC were purified to homogeneity, and BceR was purified up to 90% purity. The identities of the proteins were confirmed by trypsin digestion and liquid chromatography mass spectrometry (LC/MS), and their molecular masses were confirmed by electrospray-ionization MS at Toronto’s Sick Kids Advance Protein Technology Center (Toronto, Canada). GraR, GraRN, and GraRC are monomers in solution, as indicated by the size exclusion chromatography (Figure 1).\n\nA) The chromatographs represent the elution profile of the GraR, GraRN and GraRC proteins on a HPLC size-exclusion TSK Gel (7.8mm x 30cm, 5µm) column calibrated using the following proteins: aprotonin (6.5 kDa), carbonic anhydrase (29 kDa), ribonuclease A (37 kDa), ovalbumin (45 kDa) and conalbumin (75 kDa). B) Calibration graph of log of molecular masses against the retention time of each protein standard.\n\nCloning of graX was performed into two ways to result in production of a tagless GraX or fused to the COOH-terminus of a MAT-FLAG tag. In both cases, the proteins were purified to 90%. SDS-PAGE indicated presence of GraX dimers in solution (data not shown). Dimeric GraX species were removed in the presence of DTT, suggesting that the single cysteine residue of GraX could mediate dimerization through disulfide bond formation. Aggregation of GraX at high protein concentrations (>50 µM) hampered our efforts to determine the oligomerization state of GraX by size exclusion chromatography.\n\nProduction of tagless VraF was in good amount but in insoluble form. We fused a (His)6-tag to the COOH-terminus of VraF which increased the solubility of the produced protein and resulted in its purification at 90% homogeneity.\n\nCytoplasmic domains of histidine kinases have been used as model to study the autokinase activities of full-length proteins18,19. Herein, we undertook the study of the autokinase activities of BceS and GraS. Efforts to express full-length graS resulted in production of GraS in insoluble form.\n\nAutophosphorylation of GST-BceS in the presence of 1 mM ATP, at room temperature, showed a sharp increase in the signal intensity during the first 15 min, followed by saturation over the next 45 min (Figure 2). The pseudo-first order rate constant was determined to be 0.15 ± 0.03 min-1. In contrast, GraS did not undergo phosphorylation either as GST-GraS or as His-GraS (Figure 3A, B). We tried different ATP concentrations and different concentrations of GST-GraS or His-GraS, but no autophosphorylation activity of GraS was observed (data not shown). As a positive control in our experiments, we used GST-VraS16. In addition, we investigated the effect of the auxiliary protein GraX on the autokinase activity of GST-GraS and did not observe any effect (Figure 4).\n\n(A) GST-BceS (5 µM) was incubated with [γ-32P] ATP (1 mM) in 50 mM Tris, 50 mM KCl, 20 mM CaCl2, and 5 mM MgCl2 (pH 7.4). Reactions were quenched at different incubation times and analyzed by 12.5% SDS-PAGE. (B) The experimental data obtained in (A) were quantified using ImageJ and plotted against the incubation time (the error bars represent the standard deviations calculated from three independent experiments). The data were fitted to the equation given in the Experimental Section.\n\n(A) GST-GraS (5 µM) in 50 mM Tris, 50 mM KCl, 10 mM CaCl2, and 5 mM MgCl2 (pH 7.4) was incubated with 10 µM γ-32P-ATP. The reaction was quenched at different time intervals. (B) Autophosphorylation of His-GraS under the same conditions as in (A). Left panel represent phosphor imaging and right panels represent the Coomassie staining of the SDS-PAGE gels.\n\nGST-GraS 5 µM alone or along with 2 or 5 µM GraX was incubated at room temperature for 30 min before adding [γ-32P] ATP. GraX (5 µM) was used as control. Reactions were quenched at different time intervals and analyzed by 12.5% SDS-PAGE. On left side are shown the radiogram images of the SDS-PAGE and on the right the Coomassie stained images of the SDS-PAGE.\n\nInterestingly, we noted that presence of sodium phosphate and CaCl2 in the phosphorylation buffer resulted in false-positive phosphorylation of His-GraS or GST-GraS, and a similar observation was made for BSA (Figure 5). This could be due to the formation of insoluble calcium phosphate species in the buffer. Proper buffer exchange of the GST-GraS into Tris-HCl buffer eliminated the non-specific phosphorylation of GraS (Figure 5).\n\n(A) GST-GraS (5 µM) in the phosphorylation buffer supplemented with 10 mM CaCl2 and 10 mM sodium phosphate incubated with [γ-P32] ATP. The samples were resolved in a 12.5% SDS-PAGE. Gels were analyzed by autoradiography. (B) His-GraS (5 µM) or BSA (5 µM) was incubated in the phosphorylation buffer supplemented with different concentrations of sodium phosphate in the presence or absence of 10 mM CaCl2 and incubated with [γ-P32] ATP. Samples at a particular condition were quenched at 30 and 60 min (respectively, first and second lane of each sample) and analyzed by 12.5% SDS-PAGE. Gels were analyzed by autoradiography.\n\nThe autophosphorylation of BceS allowed us to investigate the phosphotransfer between the kinase and its cognate RR, BceR. Incubation of the phosphorylated GST-BceS (2 µM) with BceR (10 µM) resulted in the phosphotransfer of the phosphoryl group to BceR (Figure 6). The maximum amount of phosphorylated BceR was achieved within 5 min, at which point phosphorylated-BceR species started to decrease, suggesting that GST-BceS has phosphatase activity in addition to its kinase activity in analogy with the observations made for VraSR system16. Because of the high sequence homology between GraR and BceR and their similar functions, we investigated whether there was cross-talk between BceS and GraR. Incubation of phosphorylated GST-BceS with GraR did not result in the phosphorylation of GraR (data not shown).\n\nBceR (5 µM) was incubated with GST-BceS-32P (1 µM) at different time intervals in 50 mM Tris, 50 mM KCl, 20 mM CaCl2, and 5 mM MgCl2 (pH 7.4). The samples were analyzed by 15% SDS-PAGE. Gels were analyzed by autoradiography.\n\nBceR and GraR do not undergo phosphorylation by acetyl phosphate. BceR and GraR were incubated with acetyl phosphate under conditions known to phosphorylate VraR16. Samples were analyzed in a C4 reverse-phase column connected to a HPLC (Prostar, Agilent). We found that BceR and GraR did not undergo phosphorylation. Under the same conditions, VraR underwent phosphorylation (Figure 7, Figure 8), as reported before16.\n\nA 50 µL reaction was prepared containing 9.52 µM of BceR incubated in the absence (A) or presence of 50 mM of acetylphosphate in 50 mM Tris, 50 mM KCl, and 20 mM MgCl2 (B) (The smaller peak that elutes at 17 min is an impurity in both graphs A and B). The reaction mixtures were incubated at 37°C for 1 hour and then 40 µL were loaded onto an HPLC C4 reverse phase column. Elution of the protein was monitored. As a control, we monitored phosphorylation of VraR under the same conditions (C) (The smaller peak that elutes at 25.2 min corresponds to the phosphorylated VraR).\n\nFull length GraR (40 µM) was incubated with 50 mM acetyl phosphate in 50 mM Tris, 50 mM KCl, and 20 mM MgCl2 for different time intervals at 37°C. The samples were analyzed on an HPLC C4 reverse phase column by monitoring the absorbance at 212 nm (Y-axis; X-axis is the elution time in min).\n\nTo investigate the interactions between the histidine kinases and their cognate response regulators, GST-GraS or GST-BceS was immobilized onto the glutathione resin. The resin-bound GST-GraS or GST-BceS was incubated with GraR or BceR, respectively. BceR co-eluted with BceS during the elution steps (Figure 9A). By contrast, GraR eluted during the washing steps when incubated with GraS at 1:2 ratio (Figure 10A). In the case when GraR concentration was 5-fold more than GraS, we observed co-elution of GraR with GST-GraS during the elution steps (Figure 9B).\n\nThe bait proteins: GST-BceS (A), GST-GraS (B), GST-GraS (C), GST-GraSCA (D), MAT-FLAG-GraX (E, F), His-VraF (G, H), were immobilized onto their respective resins, glutathione resin (A–D) or Ni-NTA resin (E–H). The resins were washed to remove unbound proteins. The prey proteins, BceR (A), GraR (B, F), GraX (C, D, G) or GraS (H) were incubated with the resins at room temperature, and the unbound proteins were removed through seven successive washes. The protein contents of two successive elution fractions were analyzed by 12.5% SDS-PAGE. The ratios of bait protein to prey protein are given in parenthesis in each case.\n\n(A) GST-GraS was immobilized onto the glutathione resin and incubated with GraR. The resin was washed seven times, and finally eluted three times with 10 mM glutathione. Lanes: M, marker; GraS, protein prior to immobilization; GraR, protein prior to incubation with resin; In, incubation mixture in the absence of resin; U, unbound fraction; W1 and W7, fractions collected through washes of the resin; E1, E2, E3, three subsequent elution fractions collected when resin was incubated with elution buffer. (B) GST-GraS was immobilized onto the glutathione resin and incubated with BSA. Similar protocol of washing and elution was used as in (A). (C) GST-GraS was immobilized onto the glutathione resin followed by the protocol given in (A). (D) GST was immobilized onto the glutathione resin, followed by the protocol given in (A). (E) GraR was immobilized onto the Ni-NTA resin, washing and elutions were done as in (A).\n\nTo ensure the specificity of the interactions in our experimental set-up, we used BSA as the prey. In this case, BSA (at a ratio of 1:1) was not retained by the resin-bound GST-GraS. Elution with 10 mM reduced glutathione released only GST-GraS (Figure 10B).\n\nTo investigate the interaction between GraS and GraX, pull-down experiments were carried out by incubating resin-bound GST-GraS with GraX at the 1:1.3 ratio. No GraX eluted from the column during the seven washes of the resin. GST-GraS and GraX co-eluted in the first fraction of the elution step (Figure 9C). We repeated the experiments by immobilizing GST only and incubating the resin-bound GST with GraX. Using the same protocol for washing and elution, we observed that GraX eluted in the first three washes, and GST eluted alone when the resin was incubated with 10 mM glutathione buffer (Figure 10).\n\nThe full-length GraS protein consists of two domains, the N-terminal dimerization and histidine phosphotransfer domain (DHp) and the C-terminal ATP-binding domain (CA)20. The DHp domain harbors the conserved histidine residue, His129, that undergoes phosphorylation upon activation of the kinase, and the CA domain binds to ATP and catalyzes the transfer of the γ-phosphoryl group of ATP to the conserved histidine residue. To determine which GraS domain interacts with GraX, we investigated its interaction with GraSCA (181–346) and GraSDHp-CA (110–346) using the pull-down assay. In this experiment, GST-GraSCA was immobilized on the resin and incubated with GraX. At a ratio of 1:1.2 GraX:GST-GraSCA, we did not observe interaction between GraX and GST-GraSCA (Figure 9D). However, at the same ratio, GraX was pulled down by GraSDHp-CA (Figure 9E). This is a strong indication that GraX requires the DHp domain of GraS for interaction.\n\nTo investigate the interaction between GraX and GraR, MAT-FLAG-GraX was immobilized onto Ni-NTA resin. Immobilized GraX was incubated with GraR at a 1:1.2 ratio. GraR co-eluted with GraX during the elution steps (Figure 9F). GraR was not retained by the resin alone (Figure 10E).\n\nHis-VraF immobilized on Ni-NTA column was able to recruit GraX when incubated at 1:2 ratio with this protein (Figure 9G). To investigate the interaction of VraF with GraS, we looked at three different constructs of GraS: the full-length cytoplasmic domain of GraS which harbors partially the linker (residues 77–110) that connects the cytoplasmic domain to the membrane binding domain; GraSDHp-CA which lacks completely the linker; and GraSCA that harbors the ATP-binding domain. The pull down experiments showed that only full-length cytoplasmic domain of GraS was pull-downed by VraF (Figure 9H), indicating that VraF requires the linker region of GraS (77–110) for interaction.\n\nWe used CD to assess the overall folding of the target proteins and ensure that they maintain their structure during our experimental conditions. These experiments revealed that BceR and GraR share similar CD signature, indicating that any difference in their activities is not due to abnormal folding of the proteins in our experiments. GST-BceS and GST-GraS also share similar CD signature, indicating that they, too, share a similar folding pattern and any difference in their activities is not due to abnormal folding of the proteins in our experiments (Figure 11).\n\nCD spectra of GST-GraS (20 μM), His-GraS (20 μM), GraR (30 μM), GraRC (40 μM), GraRN (40 μM), BceR (20 µM), BceS (40 µM) and GraX (18 μM) were obtained in 30 mM Tris (pH 7.0) supplemented with 5 mM MgCl2. The spectra were corrected for the buffer contribution.\n\nTo confirm that GraR is a functional protein in our study, we assessed the DNA-binding activity of this protein through DNase I footprinting experiments. These experiments showed that GraR bound to PvraFG and protected a specific ~24 bp region located 110 bp upstream of the transcription starting point on the coding strand (Figure 12). On the non-coding strand, the protected region was found 114 bp upstream of the PvraFG transcription starting point (Figure 13). The protected region overlapped with the proposed GraR DNA-binding sequence15,21. We calculated the binding affinity of GraR for the target DNA (expressed as the dissociation constant Kd) as the concentration of GraR that provided 50% protection from DNase I. Intensities of four bands, in the protected region, were measured at different GraR concentrations using ImageJ software. The estimated Kd was 0.8 ± 0.2 µM. Another region of PvraFG, spanning from -109 to -88, was slightly protected by GraR at concentrations greater than 15 µM.\n\nPvraFG (10 ng) labeled on the coding strand was incubated with increasing concentrations of GraR or GraRC and subjected to DNase I. The DNA sequence protected by GraR is shown on the right. The dashed lines indicate the binding sites, and the solid lines show the palindromic sequence in DNA, as suggested by Dintner et al.21.\n\nPvraFG (10 ng) labeled on the non-coding strand was incubated with increasing concentrations of GraR or GraRC and subjected to DNase I. The DNA sequence protected by GraR is shown on the right. The dashed lines indicate the binding sites, and the solid lines show the palindromic sequence in DNA, as suggested by Dintner et al.21.\n\nGraRC protected similar regions of PvraFG as full length GraR (Figure 6); however, it protected these regions at higher GraRC concentrations, indicating that the N-terminal domain has a role in the interaction of GraR with DNA probably mediating dimerization of GraR. The estimated Kd value for GraRC was 2.4 ± 0.5 µM (Figure 14). GraR did not protect any region on the graRS promoter (Figure 14), which corroborates previous findings that GraR does not regulate expression of its operon (15) and furthermore confirms that GraR is a functional protein in our experiments.\n\nPercentage of protected DNA in the region -110- to -133 is plotted against protein concentration used in the assay. We used the intensity of four most prominent bands in the GraR-protected region as measured using Image J (NIH softeware) and averaged them out to calculate the percentage of protected DNA. Error bars indicate standard error of mean calculated from three independent experiments.\n\n\nDiscussion\n\nThe GraSR TCS, the VraFG ABC transporter and the accessory factor GraX are proposed to form a five-component system to control the response to CAMPs in S. aureus12. The ABC transporter is essential to CAMP sensing but does not play a role in the resistance to CAMPs. VraG, a permease composed of 10 transmembrane domains, has a large extracellular domain that is proposed to directly sense CAMPs and transduce intracellularly the signal through GraSR12. In addition, the accessory protein of the graXRS operon, GraX, is involved in both signaling of and resistance to CAMPs12, but its function is not known. GraS is involved in signaling and GraR is involved in CAMPs resistance by directly regulating the genes that are responsible for D-alanylation of wall teichoic acid and lysinylation of phosphatidyl-glycerol within the cell membrane6,9,10.\n\nSince GraS and GraR constitute a TCS, it is supposed that upon intercepting a signal the GraS kinase will be activated through a trans-autophosphorylation process, whereby a conserved histidine residue of GraS will undergo phosphorylation. This signal will presumably be further transduced intracellularly through a second phosphorylation step, whereby the phosphoryl group of GraS will be transferred to GraR, resulting in its activation and initiation of the downstream regulatory steps. GraS lacks a bona-fide extracellular sensor domain, and this is considered as evidence that GraS is not directly involved in sensing CAMPs22,23, however, mutations at the extracellular loop that connects that two transmembrane helices of GraS have been reported to affect CAMP resistance8,10, suggesting otherwise.\n\nThe cytoplasmic domains of histidine kinases have been considered good models to study the autokinase/kinase activities of HKs18. We show that the cytoplasmic domain of GraS does not have autophosphorylation activity. By contrast, a similar histidine kinase, BceS, undergoes autophosphorylation. Similar regions of GraS and BceS were cloned guided by their sequence homology (Figure S1). Their CD spectra showed that both proteins share the same folding (Figure 11). Hence, any difference between these two proteins in terms of their autophosphorylation activities should be considered an indication of their differences in function, only, and not misfolding of GraS. This begs the question, if GraS does not have autophosphorylation activity and it is not the CAMP sensor, how is the signal transduced through GraSR?\n\nOur study shows that BceSR behaves as a typical TCS, i.e. BceS has autophosphorylation activity and it phosphorylates its cognate RR. Similarly to GraSR, BceSR also depends on an ABC transporter, BceAB, for sensing bacitracin, but unlike GraSR no auxiliary protein is required for signal transduction through BceSR14. The dependence of CAMP signaling on the accessory protein GraX and our findings that GraX requires the DHp domain of GraS for interaction and it interacts with VraF, and in turn VraF requires the linker region of GraS for interaction, suggest that both these proteins may be involved in the activation of GraS kinase activity.\n\nOur study shows that GraS interacts weakly with GraR, by contrast, BceS interacts strongly with BceR. The latter observation indicates that in a typical TCS, where HK phosphorylates its cognate RR, the unphosphorylated HK interacts with its cognate RR and that lack of interaction between GraR and GraS may be due to an improper alignment of structural elements in the DHp region known to determine the interaction between HK and its cognate RR24. Notably, both GraR and BceR, and GraS and BceS share very similar CD signatures removing any doubt that any difference in function between these pairs is due to misfolding of GraR and GraS.\n\nIn vivo studies showed that deletion of graS prevents the S. aureus response to CAMPs12,15, which suggests that GraS plays a role in the regulation of GraR activity. This role of GraS is further confirmed by our finding that GraR does not undergo phosphorylation by acetyl phosphate, ruling out that this small-molecule phosphate donor could phosphorylate GraR in vivo. Notably, the interaction of GraX with GraS/R may result in increased local concentration of GraR, which in turn may forge interaction between GraS and GraR. Further, the dimeric state of GraX observed in our study may accommodate the observed interactions of GraX with GraS, GraR and VraF suggesting that GraX may serve as a scaffold, bringing several partners in close proximity.\n\nThe formation of a potential complex among GraS, GraX, GraR, and VraF may also serve to regulate the CAMPs signal transduction process. The linker that connects the two transmembrane-spanning α-helices of GraS is believed to reside within the lipid bilayer membrane of the cell due to its short length (nine amino acids) and as such, it may only be able to detect stimuli from within or at the membrane22. However, because the conditions in and around the membrane constantly change due to cell growth and trafficking on both sides, it might be challenging for the GraS membrane-embedded linker to reliably sense the signal and initiate the signaling process. The association of GraS with the ABC transporter, which possesses a longer extracellular linker, offers the kinase an accurate reading of the signal among many look-alike signals. This hypothesis is supported by the report that GraSR-VraFG responds to selective CAMPs11. Likewise, BraSR-BraDE and BceSR-BceAB respond to selective molecules. In contrast, VraSR and LiaSR TCSs, respectively in S. aureus and B. subtilis, which are not dependent on an ABC transporter for signaling, respond to cell wall damage caused by different classes of antibiotics that target cell wall biosynthesis14,25.\n\nIn conclusion, our study shows that the cytoplasmic domain of GraS does not have autophosphorylation activity, unlike that of BceS, and there is a weak interaction between GraS and its cognate response regulator GraR. These observations suggest that GraSR may not support a signal transduction process on its own. The tight interactions of GraX with GraS, GraR, and VraF suggest that GraX may serve as a scaffold, where VraF, GraS, and GraR dock to increase the local concentration of proteins and forge further interactions among them. This complex formation may enable phosphorylation of GraS. Upon activation of GraS, the extracellular signal may be transduced to GraR through a phosphotransfer process, leading to the initiation of downstream events.\n\n\nData availability\n\nF1000Research: Dataset 1. Data for signal transduction mechanism of GraSR in Staphylococcus aureus, 10.5256/f1000research.5512.d3722826", "appendix": "Author contributions\n\n\n\nUzma Muzmal, Daniel Gomez and Fenika Kapadia performed the experiments and analyzed the data. Dasantila Golemi-Kotra designed the project, the experiments, analyzed the data and wrote the manuscript. All authors agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by a Discovery grant to DGK from the Natural Sciences and Engineering Research Council of Canada and an Early Researcher Award to DGK from Ontario Ministry of Economic Development and Innovation.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\n\n\n\nReferences\n\nLowy FD: Staphylococcus aureus infections. N Engl J Med. 1998; 339(8): 520–532. PubMed Abstract \| Publisher Full Text\n\nArcher G L: Staphylococcus aureus: a well-armed pathogen. Clin Infect Dis. 1998; 26(5): 1179–1181. PubMed Abstract \| Publisher Full Text\n\nIppolito G, Leone S, Lauria FN, et al.: Methicillin-resistant Staphylococcus aureus: the superbug. Int J Infect Dis. 2010; 14(Suppl 4): S7–11. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFalord M, Karimova G, Hiron A, et al.: GraXSR proteins interact with the VraFG ABC transporter to form a five-component system required for cationic antimicrobial peptide sensing and resistance in Staphylococcus aureus. Antimicrob Agents Chemother. 2012; 56(2): 1047–1058. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStock AM, Robinson VL, Goudreau PN: Two-component signal transduction. Annu Rev Biochem. 2000; 69: 183–215. PubMed Abstract \| Publisher Full Text\n\nRietkotter E, Hoyer D, Mascher T: Bacitracin sensing in Bacillus subtilis. Mol Microbiol. 2008; 68(3): 768–785. PubMed Abstract \| Publisher Full Text\n\nFalord M, Mader U, Hiron A, et al.: Investigation of the Staphylococcus aureus GraSR regulon reveals novel links to virulence, stress response and cell wall signal transduction pathways. PLoS One. 2011; 6(7): e21323. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBelcheva A, Golemi-Kotra D: A close-up view of the VraSR two-component system. A mediator of Staphylococcus aureus response to cell wall damage. J Biol Chem. 2008; 283(18): 12354–12364. PubMed Abstract \| Publisher Full Text\n\nBelcheva A, Verma V, Golemi-Kotra D: DNA-binding activity of the vancomycin resistance associated regulator protein VraR and the role of phosphorylation in transcriptional regulation of the vraSR operon. Biochemistry. 2009; 48(24): 5592–5601. PubMed Abstract \| Publisher Full Text\n\nYamamoto K, Hirao K, Oshima T, et al.: Functional characterization in vitro of all two-component signal transduction systems from Escherichia coli. J Biol Chem. 2005; 280(2): 1448–1456. PubMed Abstract \| Publisher Full Text\n\nSkerker JM, Prasol MS, Perchuk BS, et al.: Two-component signal transduction pathways regulating growth and cell cycle progression in a bacterium: a system-level analysis. PLoS Biol. 2005; 3(10): e334. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrell T, Lacal J, Busch A, et al.: Bacterial sensor kinases: diversity in the recognition of environmental signals. Annu Rev Microbiol. 2010; 64: 539–559. PubMed Abstract \| Publisher Full Text\n\nDintner S, Staron A, Berchtold E, et al.: Coevolution of ABC transporters and two-component regulatory systems as resistance modules against antimicrobial peptides in Firmicutes Bacteria. J Bacteriol. 2011; 193(15): 3851–3862. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMascher T: Intramembrane-sensing histidine kinases: a new family of cell envelope stress sensors in Firmicutes bacteria. FEMS Microbiol Lett. 2006; 264(2): 133–144. PubMed Abstract \| Publisher Full Text\n\nHiron A, Falord M, Valle J, et al.: Bacitracin and nisin resistance in Staphylococcus aureus: a novel pathway involving the BraS/BraR two-component system (SA2417/SA2418) and both the BraD/BraE and VraD/VraE ABC transporters. Mol Microbiol. 2011; 81(3): 602–622. PubMed Abstract \| Publisher Full Text\n\nSkerker JM, Perchuk BS, Siryaporn A, et al.: Rewiring the specificity of two-component signal transduction systems. Cell. 2008; 133(6): 1043–1054. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKuroda M, Kuroda H, Oshima T, et al.: Two-component system VraSR positively modulates the regulation of cell-wall biosynthesis pathway in Staphylococcus aureus. Mol Microbiol. 2003; 49(3): 807–821. PubMed Abstract \| Publisher Full Text\n\nMuzamal U, Gomez D, Kapadia F, et al.: Data for signal transduction mechanism of GraSR in Staphylococcus aureus. F1000Research. 2014. Data Source" }	[ { "id": "6514", "date": "31 Oct 2014", "name": "Ambrose Cheung", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a biochemical study that dissects the interaction of a two component regulatory system (GraRS) with a third component (GraX) and its downstream ABC transporter system composed of a permease (vraG) and an ATPase (VraF). The major contribution of this paper is to ascertain the interaction of GraX with VraF and GraS. In addition, VraF also interacts with GraS, thus implying a complex system of interaction likely involving at least four proteins. However, the enthusiasm for this manuscript is tempered by several major deficiencies that need to be amended.Major issues:The comparison of BceRS to GraRS is invalid because the correct comparison should be between BraRS (SA2417-2418) and BceRS. Similar to BceRS, BraRS is a TCRS that, together with BraDE (efflux pump system), is required for bacitracin resistance in S. aureus (ref. 23). The interaction of different protein depends on pulldown study where it appears that there is likely to be protein overload to the system (e.g. GraR appears in large amount of the unbound fraction). This reviewer believes the amount of protein used as binding partner should be reduced (to avoid non-specific interaction) and then detected by immunoblots. The authors totally ignored the contribution of VraG The critical protein binding studies in Fig. 9 should be displayed as in Fig. 10 where data on unbound, washed and eluted fractions should be presented. Also, control protein should be included in these data ensure there is no non-specific protein interaction, especially when proteins are used at a relatively high quantities. It would be useful to confirm different protein binding studies (Fig. 9) with another method such as Biacore, FTIR or two-hybrid system. Cull back on negative data and mention in the text. The authors should consider using phosphorylated GraR on the DNAse I footprinting assay. Minor points:ABC transporter senses CAMP (page 3) – this is more controversial. I am not sure how the transporter senses since this is not a typical sensing system, especially when this is an efflux system with no ligand binding protein exposed to the outside. What is the control for Figure 3 in this system? In Fig. 7C, what is the elution profile of the “unphosphorylated VraR”? There appears to be mislabeling in Fig. 9G (GraX instead of GraS). Similarly, Fig. 10A is also mislabeled (GraR instead of GraX). Explain the rationale for the different protein ratios in the protein binding experiments. On p.13, third paragraph, GraRc protected similar….. (Fig. 6). The figure is wrongly references. In the discussion, the author mentions that “GraS lacks a bona-fide extracellular sensor domain”. There is a short extracellular loop in GraS. How long does it have to be to be “bona fide”? Could binding of GraS by CAMP activate phosphorylation?", "responses": [ { "c_id": "1071", "date": "10 Nov 2014", "name": "Dasantila Golemi-Kotra", "role": "Author Response", "response": "Thank you very much for your careful review of our paper. I have addressed your concerns in the order they were raised.Major issues:\"The comparison of BceRS to GraRS is invalid because the correct comparison should be between BraRS (SA2417-2418) and BceRS. Similar to BceRS, BraRS is a TCRS that, together with BraDE (efflux pump system), is required for bacitracin resistance in S. aureus (ref. 23).\"It is important to note that GraSR proteins share the highest sequence identities with BceSR proteins, rather BraSR proteins, and that is why for the purpose of characterization of GraSR activities we refereed to this two-component system, rather than BraSR. \"The interaction of different protein depends on pulldown study where it appears that there is likely to be protein overload to the system (e.g. GraR appears in large amount of the unbound fraction). This reviewer believes the amount of protein used as binding partner should be reduced (to avoid non-specific interaction) and then detected by immunoblots.\"This is a valid concern. I would like to make the point, as to clarify our experimental conditions and remove any misunderstanding, the protein concentration for the “bait” does not exceed 10-15 µM and the protein concentration for the “prey” is kept between 10-30 µM (1:1 or 1:2 ratio), with the exception when we did not see interaction under these conditions and we had to increase concentration of the “prey” protein to 5-fold more than the “bait” protein (the case of GraS:GraR). These protein concentrations are actually at the low end of any other technique used for binding experiments. In addition, as our data show, under our experimental conditions GraR does not bind to GraS at 2:1 ratio and GraX and VraF do not interact with all the GraS constructs. As well, the control experiments demonstrate that under our working conditions there are no non-specific interactions. \"The authors totally ignored the contribution of VraG\"VraG is an important player in the signal transduction process of GraSR. It is a membrane embedded protein that is not amenable to the methods used in our study. \"The critical protein binding studies in Fig. 9 should be displayed as in Fig. 10 where data on unbound, washed and eluted fractions should be presented. Also, control protein should be included in these data ensure there is no non-specific protein interaction, especially when proteins are used at a relatively high quantities.\"We believe that Figure 10 addresses the issues of non-specificity that may arise from these experiments and addition of other figures will be redundant. \"It would be useful to confirm different protein binding studies (Fig. 9) with another method such as Biacore, FTIR or two-hybrid system.\"We have tried isothermal titration calorimetry experiments. But this method, like FTIR and Biacore, suffers from the need of having to use higher protein concentrations in the assay which leads to aggregation of our target proteins. \"Cull back on negative data and mention in the text.\"It has been the Journal’s advice to include the data wherever possible and we followed their advice. \"The authors should consider using phosphorylated GraR on the DNAse I footprinting assay.\"We show that GraR does not undergo phosphorylation by acetyl phosphate, Figure 8.Minor points:\"ABC transporter senses CAMP (page 3) – this is more controversial. I am not sure how the transporter senses since this is not a typical sensing system, especially when this is an efflux system with no ligand binding protein exposed to the outside.\"The participation of the ABC transporters in signaling is a new model that has been proposed recently (Ref. 21 and 22). Hence, we have presented in our article both views that are currently present in the literature, that of GraS serving as the sensor (Ref. 8,11) and that of the extracellular region of VraG serving as the sensor (Ref. 12). \"What is the control for Figure 3 in this system?\"Figure 2 is the control for this experiment as both proteins were subjected under the same phosphorylation conditions. \"In Fig. 7C, what is the elution profile of the “unphosphorylated VraR”?\"We have published these data in Ref 18 (Supplemental data). \"There appears to be mislabeling in Fig. 9G (GraX instead of GraS). Similarly, Fig. 10A is also mislabeled (GraR instead of GraX).\"Thank you for noting them. We have accordingly done the corrections. \"Explain the rationale for the different protein ratios in the protein binding experiments.\" The slight deviations on the protein ratios from 1:1 ratio in the pull-down experiments was due the actual stock protein concentrations that were available after protein purification/concentration. Some of these proteins are prone to aggregation at relatively low concentrations e.g. 30-50 µM, such as GraS and GraX, and/or not produced at large quantities such as VraF. \"On p.13, third paragraph, GraRc protected similar….. (Fig. 6). The figure is wrongly references.\"Corrected accordingly. \"In the discussion, the author mentions that “GraS lacks a bona-fide extracellular sensor domain”. There is a short extracellular loop in GraS. How long does it have to be to be “bona fide”?\"This is a good question. There is a discussion on this matter in literature (Ref. 22). There is still room to further investigate this matter. \"Could binding of GraS by CAMP activate phosphorylation?\"There are studies that indicate that GraS is directly involved in sensing of CAMPs (Ref. 8, 11), which cannot be ignored. Our work is ongoing to further explore the signaling mechanism by GraS." } ] }, { "id": "6605", "date": "10 Nov 2014", "name": "Jonathan D. Partridge", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe work by Muzamal and co-workers is an in depth study of the Staphylococcus two-component system GraRS, its auxiliary protein GraX and associated ABC transporter system vraG. The work is thorough and shows a methodical approach to characterizing the players in this system, however a few issues need to be addressed. Major comments:Considering how detailed the work is, it is surprising that VraG, co-transcribed with VraF and under the regulatory control of GraR, is not investigated. Why? If components of this system sense and bind CAMPs (page 3) to effect a change, perhaps some of these binding studies should have been done in the presence as well as absence of CAMPs. Minor comments: pg. 4, Protein concentrations determined by Bradford assay, a reference should be included. pg. 5, \"GraX fractions were pooled and concentrated\". How were proteins concentrated? pg. 5, cell cultures were cooled to 4°C before IPTG induction at 16°C for 16 h. Why? pg. 6, DNase footprinting experiments, +28 to -168 was used, what was the justification? Has the promoter architecture been mapped to solely this region? If so, a reference should be added. pg. 7, \"cytoplasmic domains of histidine kinases have been used as model to study...\", should be “models”. Why does the ratio of protein:protein used in experiments vary? The quality of some of the gel figures are perfectly clear whilst some are poor, exacerbated when the document is printed. More consistency in quality would improve interpretation of the manuscript. Due to the large number of expression vectors used in the study, a small table should be added to note the designation, host, resistance, induction etc, of those plasmids used and those plasmids made for reader clarity and reference. Some of the figures referred to in the manuscript do not match to the correct figures, this should be thoroughly checked.", "responses": [ { "c_id": "1070", "date": "10 Nov 2014", "name": "Dasantila Golemi-Kotra", "role": "Author Response", "response": "Thank you very much for the critical reading of our work. We have addressed your comments below. Major comments:\"Considering how detailed the work is, it is surprising that VraG, co-transcribed with VraF and under the regulatory control of GraR, is not investigated. Why?\"VraG is a membrane embedded protein with 10 membrane spanning a-helices. Hence this protein is not amenable to our experiments. \"If components of this system sense and bind CAMPs (page 3) to effect a change, perhaps some of these binding studies should have been done in the presence as well as absence of CAMPs.\"This is a good point. Our GraS construct does not carry the membrane bound domain and the other proteins are cytoplasmic proteins. The ABC transporter VraFG is shown not to be a transporter for the CAMPS. Nonetheless, we included indolicidin in the phosphorylation experiments for GraS and GraR as well as DNA-binding experiments with GraR. We did not see any affect. These data are not shown and not discussed as they do not provide any important insight to this study.Minor comments:\"pg. 4, Protein concentrations determined by Bradford assay, a reference should be included.\"We used the Bradford reagent obtained from GE Healthcare, and used the manufacturer’s instructions for its use. The text is corrected to reflect this fact. \"pg. 5, \"GraX fractions were pooled and concentrated\". How were proteins concentrated?\"This information is added to the main text. We use an Amicon Stirred Cell. \"pg. 5, cell cultures were cooled to 4°C before IPTG induction at 16°C for 16 h. Why?\"Under this conditions, we obtained an optimum production of the soluble protein. This could be due to the decrease of gene expression rate at the low temperature, which may result in a smaller chance of newly synthesized protein to be trapped as inclusion bodies. \"pg. 6, DNase footprinting experiments, +28 to -168 was used, what was the justification? Has the promoter architecture been mapped to solely this region? If so, a reference should be added.\"We used the entire intergenic region between the graXSR operon and the upstream operon in S. aureus Mu 50 genome, and part of the coding region of graXSR operon. Earlier studies on the proposed GraR binding sequence are provided in the text, ref. 15 and 21 (page 6 and 13). \"pg. 7, \"cytoplasmic domains of histidine kinases have been used as model to study...\", should be “models”.\"This is corrected as indicated. \"Why does the ratio of protein:protein used in experiments vary?\"Stock solutions for each protein were different. \"The quality of some of the gel figures are perfectly clear whilst some are poor, exacerbated when the document is printed. More consistency in quality would improve interpretation of the manuscript.\"We regret the apparent inconsistency in figure quality. This is due to the protective measurements that we need to take when taking pictures of radioactive SDS-PAGE that are stained with Coomassie (in order to see the protein bands); we have to use a plastic wrap. \"Due to the large number of expression vectors used in the study, a small table should be added to note the designation, host, resistance, induction etc, of those plasmids used and those plasmids made for reader clarity and reference.\"We have used only three expression vectors: pET26, pGEX-4T and pT7-MAT-tag-Flag1. \"Some of the figures referred to in the manuscript do not match to the correct figures, this should be thoroughly checked.\"We have made the corrections wherever necessary." } ] } ]	1	https://f1000research.com/articles/3-252
https://f1000research.com/articles/3-110/v1	14 May 14	{ "type": "Research Article", "title": "Readable workflows need simple data", "authors": [ "Claas-Thido Pfaff", "Karin Nadrowski", "Sophia Ratcliffe", "Christian Wirth", "Helge Bruelheide", "Karin Nadrowski", "Sophia Ratcliffe", "Christian Wirth", "Helge Bruelheide" ], "abstract": "Sharing scientific analyses via workflows has great potential to improve the reproducibility of science as well as communicating research results. This is particularly useful for trans-disciplinary research fields such as biodiversity - ecosystem functioning (BEF), where syntheses need to merge data ranging from genes to the biosphere. Here we argue that enabling simplicity in the very beginning of workflows, at the point of data description and merging, offers huge potentials in reducing workflow complexity and in fostering data and workflow reuse. We illustrate our points using a typical analysis in BEF research, the aggregation of carbon pools in a forest ecosystem. We introduce indicators for the complexity of workflow components including data sources. We show that workflow complexity decreases exponentially during the course of the analysis and that simple text-based measures help to identify bottlenecks in a workflow and group workflow components according to tasks. We thus suggest that focusing on simplifying steps of data aggregation and imputation will greatly improve workflow readability and thus reproducibility. Providing feedback to data providers about the complexity of their datasets may help to produce better focused data that can be used more easily in further studies. At the same time, providing feedback about the complexity of workflow components may help to exchange shorter and simpler workflows for easier reuse. Additionally, identifying repetitive tasks informs software development in providing automated solutions. We discuss current initiatives in software and script development that implement quality control for simplicity and social tools of script valuation. Taken together we argue that focusing on simplifying data sources and workflow components will improve and accelerate data and workflow reuse and simplify the reproducibility of data-driven science.", "keywords": [ "Interdisciplinary approaches", "new tools and technologies", "and the increasing availability of online accessible data have changed the way researchers pose questions and perform analyses1. Workflow software enables access to distributed web services providing data1", "and enables automation of the repetitive tasks that occur in every scientific analysis. Workflow tools such as Kepler or Pegasus help to break down complex tasks into smaller pieces2", "3. However", "an increase in the complexity of analyses and datasets packed into workflows can render them difficult to understand and to reuse. This is particularly true for the “long tail” of big data4", "consisting of small and highly heterogeneous files that don’t result from automated loggers but from scientific experiments", "observations", "or interviews. The difficulty in reusing workflows and research data is not only a waste of time", "money and effort but also represents a threat to the basic scientific principle of reproducibility. Current literature on workflows deals with different tools to create and manipulate workflows5–7 as well as to keep track of data provenance2 and how semantics can be integrated into workflows8. However", "there is a lack of papers that discuss workflow components within an analysis including data processing." ], "content": "Introduction\n\nInterdisciplinary approaches, new tools and technologies, and the increasing availability of online accessible data have changed the way researchers pose questions and perform analyses1. Workflow software enables access to distributed web services providing data1, and enables automation of the repetitive tasks that occur in every scientific analysis. Workflow tools such as Kepler or Pegasus help to break down complex tasks into smaller pieces2,3. However, an increase in the complexity of analyses and datasets packed into workflows can render them difficult to understand and to reuse. This is particularly true for the “long tail” of big data4, consisting of small and highly heterogeneous files that don’t result from automated loggers but from scientific experiments, observations, or interviews. The difficulty in reusing workflows and research data is not only a waste of time, money and effort but also represents a threat to the basic scientific principle of reproducibility. Current literature on workflows deals with different tools to create and manipulate workflows5–7 as well as to keep track of data provenance2 and how semantics can be integrated into workflows8. However, there is a lack of papers that discuss workflow components within an analysis including data processing.\n\nIn the following we 1) introduce the concepts of workflow component complexity and identity as well as data complexity. We then use 2) a workflow from the research domain of biodiversity-ecosystem functioning (BEF) to illustrate these concepts. The analysis combines small and heterogeneous datasets from different working groups to quantify the effect of biodiversity and stand age on carbon stocks in a subtropical forest. In the third and last part of the paper we 3) discuss the opportunities for quantifying the complexity and identity of workflow components and data for developing useful features of data sharing platforms and fostering scientific reproducibility. In particular, we are convinced that simplicity and a clear focus are the key to adequate reuse and finally to the reproducibility of science. We use our findings to illustrate bottlenecks and opportunities for data sharing and the implementation and reuse of scientific workflows.\n\nWorkflows consist of components that communicate with each other. Data can be assembled from different sources and different techniques can be used to analyse the data. Components perform anything from simple data import and transformation tasks to the execution of complex statistical scripts or calls to remotely running data manipulation or information retrieval services2. The complexity of software or code in workflow components increases with the number of linearly independent paths9. Thus the complexity increases with every decision of a programmer or analyst that is introduced by an if-else or case statement. However, it is our experience as data managers and researchers in biodiversity sciences that most workflows shared between researchers do not include such if-else statements but contain one single path only. The Code Climate initiative provides code complexity feedback to programmers for many different programming languages https://codeclimate.com/?v=b. Their complexity measures include the number of lines used for methods as well as the repetition of identical code lines.\n\nQuantifying workflow complexity along the sequence of components may help to identify parts of workflows that need simplification. Workflows often begin with a series of steps that contain data preparation, merging and imputation. These first steps can make up to 70% of the whole workflow10.\n\nIn10, they identify common motifs in workflows including data and workflow oriented motifs. Identifying common and recurring tasks or motifs in workflows may allow for an improved sharing of code snippets and workflow components. There are many examples of sharing code snippets, including (e.g gist, stackoverflow). Providing quantitative complexity measures together with automated tagging may further increase component and data reuse. Identification of tasks may also support the use of semantic tools in workflow creation8,11.\n\nQuantifying data complexity is not as straight forward as workflow component complexity. Datasets used for synthesis in research collaborations often consist of \"dark\" data, lacking sufficient metadata for reuse4,12,13. Here we suggest that data complexity can be quantified by looking at the workflow components needed to aggregate and focus the data for analysis. One of the paradigms of data-driven science is that the analysis should be accompanied by it’s data. We argue that at the same time, data should be accompanied by workflows that offer meaningful aggregation of the data. Data complexity could then be measured by the complexity of their workflows.\n\nIn our experience as data managers of research collaborations, many datasets contain a complete representation of a certain study and thus allow us to answer more than just one single question. This is due to a \"space efficient\" use of sheets of papers and computer screens during the field period of the study. Thus, many data columns are used for different measurements, color is used to code for study sites without explicitly naming them in a separate column which constitutes bad quality data management. Later in the process of writing up, each analysis makes use of a subset of the data only. Thus, data needs to be transformed, imputed, aggregated, or merged with data from other columns to be used in an analysis12. Thus, not only the metadata but also the data columns in datasets differ in their quality and usage in a workflow.\n\nTo date, we lack a suitable feedback mechanism for data providers about the quality and re-usability of their data14. Such feedback could potentially lead to more simple and focused datasets and thus to more focused workflows that can be shared and reused more efficiently. Focused workflow components have the potential to be used as basic building blocks in a semantically guided way of workflow creation8 or to be targets of automation.\n\nIn the following we illustrate the concepts mentioned above within a typical BEF workflow. The workflow combines datasets from different working groups to assess the influence of diversity and stand age on the carbon pool in a subtropical forest. We analyse the complexity and the identity of workflow components as well as the data sources.\n\n\nThe effect of biodiversity on subtropical carbon stocks\n\nOur workflow performs a representative analysis in BEF. It aggregates carbon biomass from different pools of the ecosystem and compares plots along a gradient of biodiversity. The workflow combines data from 8 datasets to perform a linear regression model on the effect of biodiversity on carbon stocks in a subtropical forest. It takes into account carbon pools from soil, litter, woody debris, herb layer plants and trees and shrubs surpassing 3 cm diameter at breast height measured in the years 2008 and early 2009.\n\nThe data was collected by 7 independent projects of the biodiversity - ecosystem functioning - China (BEF-China) research group funded by the German Research Foundation (DFG, FOR 891). The BEF-China research group (www.bef-china.de) uses two main research platforms. An experimental forest diversity gradient of 50 ha, and 27 observational plots of 30×30 m each located in the province of Gutianshan China. The plots are situated in the Nature Reserve of Gutianshan. The observational plots were selected according to a crossed sampling design along tree species richness and stand age. The data for the workflow on carbon pools stems from observational plots spanning a gradient from 22 to 116 years consisting of 14 to 35 species15.\n\nBEF-China uses the BEFdata platform12, https://github.com/befdata/befdata) for managing and distributing data which also offers an Ecological-Metadata-Language (EML) export. We used the portal to retrieve the data and the according EML files which then were used to import the data into the Kepler Workflow system2 for analysis (Figure 1).\n\nOn the right side the opened metadata window which displays all the additional information available for the dataset.\n\nAs the underlying analysis of the workflow continues in the projects we only provide a short insight in the still preliminary findings here. The carbon pool in the observational plots ranged from 5321.18 kg to 51,095.95 kg. The linear model revealed both species richness as well as stand age increased the carbon pool. In addition, there was a significant interaction between stand age and species richness in that the increase of carbon with stand age was less steep in plots with higher species richness (p-values for stand age: 0.0006, species richness: 0.0568 and their interaction: 0.0236).\n\nWe use the Kepler workflow system (version 2.4) to build our workflow. The components in Kepler fall into two categories: “actors”, which handle all kinds of data related tasks, and “directors”, which direct the execution of components in the workflow. The components in Kepler can “talk” to each other via a port system. Output ports of components hand over their data to input ports of another component2.\n\nThe “SDF” director was used to execute our workflow as it handles sequential workflows. The data was imported into Kepler using the “eml2dataset” actor. This actor can import datasets along the conventions of the Ecological Metadata Language16. The component reads the information available in the metadata file and uses it to automatically set up output ports to allow a direct consumption of the related data by other components in the workflow. The data in the underlying carbon stock analysis is manipulated mainly by using the rich statistics environment R17. From within Kepler we use the “RExpression” actor that offers an interface to R. We aimed at a uniform and low complexity for each workflow component. As a rule of thumb we set a limit of 5 lines of code per component.\n\nTo quantify the component complexity we used the number of code lines (loc), the number of R commands (cc) and R packages (pc) used, as well as the number of input and output ports (cp) of the component (equation 1). We further calculated a relative component complexity as the ratio of absolute complexity to total workflow complexity given by the sum of all component complexities (equation 2).\n\n\n\n\n\nAs each component in the workflow starts its operation only if all input port variables have arrived, the longest port connection of a component back to a data source defines its absolute position in the workflow sequence (Figure 2). We could thus explore total workflow complexity, individual component complexities, the number of components, and the number of identical tasks (see below) along the sequence of the workflow. For this we used linear models and compared them using Akaike Information Criteria (AIC)18.\n\nThe absolute position in a workflow is defined by the distance back to the data source. The numbers on the components display the distance count back to the data source. The assignment of positions starts with 0.\n\nBased on our analysis, we classified workflow components into 12 tasks a priori (Table 1). We then used text mining tools to characterize the components automatically. For this we used the presence/absence of R commands and libraries as qualitative values, the number of input and output ports, the number of datasets a component is connected with, as well as the count of code lines. This allowed us to match the a priori tasks with the automatically gathered characteristics. We used non metric multidimensional scaling (NMDS)19 to find the two main axes of variation in the multidimensional space defined by the characteristics. We then performed linear regression to identify which of the characteristics and which of the a-priori tasks could explain variation of the two NMDS axes. We could further compare task complexities. For this we used a Kruskal-Wallis test and a post-hoc Wilcoxon test, since residuals where not normally distributed (Shapiro-Wilk test).\n\nData for the workflow comes from several data sources, differing in the number of columns as well as the number of processing steps needed within the workflow. We here introduce two measures of data column usage, one relative to the data source and one relative to the number of workflow components processing the data. We further introduce a quality measure of a data column by identifying a critical component within the workflow that signifies the actual analyses that answers our scientific question. We thus have workflow components that prepare data for the analysis and we have (few) workflow components that consume data for the analyses (Figure 3).\n\nThe components marked with a P represent preparation steps of a variable. Here we see three preparation steps so the quality is 4. The components marked with C and I represent direct consumption and an indirect influence. Those together build the variable usage together with all following influenced components.\n\nAs explained above, output ports of a data source in the workflow directly relate to data columns in the data set. Thus, the number of available ports of a data source is the “width” of a dataset, or the number of data columns. Thus, the usage of a data column in relation to the data source was calculated as the ratio of ports actually used in the workflow to the ports that were not used. This allowed us to relate the number of unused ports to the number of available ports of a data source.\n\nIn contrast, the usage of a data column in relation to the workflow is quantified by the total number of workflow components processing the data, before and after the actual analysis (equation 3). Similarly, the quality of a data column in relation to the workflow is quantified by the number of workflow components before the critical analysis, including this workflow component itself (equation 4). Thus, the higher the quality value, the lower the column’s quality. We can now compare datasets based to the usage and quality of their data as it is processed in the workflow. We did this using Kruskal-Wallis and post-hoc Wilcoxon tests.\n\n\n\n\n\nThe workflow analysing carbon pools on a gradient of biodiversity consisted of 71 components in 16 workflow positions consuming the data of 8 datasets (Table 2). The data in the workflow was manipulated via 234 lines of R code. The number of code lines per component ranged between 1 (e.g component plot_2_numeric) and 23 (component impute_missing_tree_heights) with an overall mean of 3.3 (± 3.98 SD). See Figure 3 for a graphical representation of the workflow.\n\nAlthough we aimed to keep the components streamlined and simple, the absolute and relative component complexity varied markedly. The absolute complexity ranged between 4 and 41 with an overall mean of 9.25 (± 6.77 SD), (Summary: Min. 4.0, 1st Qu. 4.0, Median 8.0, Mean 9.2, 3rd Qu. 12.0, Max. 41.0). Relative component complexity ranged between 0.69% (e.g component calculate_carbon_mass_from_biomass) and 7.03% (e.g component: add_missing_height _-broken_trees) with an overall mean of 1.59 (± 1.16 SD), (Summary: Min. 0.69, 1st Qu. 0.69, Median 1.37, Mean 1.59, 3rd Qu. 2.05, Max. 7.03).\n\nTotal workflow complexity decreased exponentially from the beginning to the end (Figure 4). The exponential decrease means that the decrease in complexity is steeper in the beginning of the workflow than at the end of the workflow, showing that complexity at the end of the workflow did not differ as much as at the beginning of the workflow. From the three models relating the sum of relative component complexities to workflow position, the one including position as logarithm (AIC = 64.69) was preferred over the one including a linear and a quadratic term for position (AIC = 65.67, delta AIC = 0.71), and the one including position as linear term only (AIC = 72.26, delta AIC = 7.3).\n\nThis figure shows the model back transformed to the original workflow positions. The gray shading displays the standard error.\n\nAt the same time, relative complexity increased in the course of the analysis (Figure 5), since our model with an intercept and linear term for workflow position (AIC = 325.85) was preferred. However, we will argue later, that this increase was mainly due to a group of workflow components of extreme simplicity at the very beginning of data import, visible in the bottom left of Figure 5. These workflow components convert text columns into numeric columns in the “data type transformation” task. As we will outline later, we took this as an opportunity to program a feature for our data portal, to convert columns mixed with text and numbers to numeric columns for the EML output.\n\nThe points are slightly jittered to handle over plotting. At each position in the workflow there are components of different type and complexity. Linear model with positions as predictor for relative components complexity. R-squared: 0.09, F-statistic: 6.57 on 1 and 61 DF, p-value: 0.01285.\n\nWe could group workflow components according to their a priori assigned tasks using text mining. The non metric multidimensional scaling had a stress value of 0.17 using 2 main axes of variation. Several of the parameters, including specific commands of R code, were correlated to axes scores (Table 3). Our a priori defined tasks could be significantly separated in the parameter space (r2 0.58, p-value 0.001): the first axis spans between the workflow tasks “data aggregation” and “modify a vector” while the second one spans between the tasks “data extraction” and “data type transformation” (Figure 6).\n\nSignif. codes: 0 ‘*’ 0.001 ‘’ 0.01 ‘’ 0.05 ‘.’ 0.1 ‘ ’ 1. P-values based on 999 permutations.\n\nThe scaling was created using the R package vegan with the Bray-Curtis distance. The large labels represent the workflow tasks. The smaller text annotations represent the characteristics used. They are slightly jittered by a factor of 0.2 horizontally and vertically to handle over plotting.\n\nOur workflow tasks had similar complexity, with only one exception: the task “data type transformation” was less complex (Kruskal-Wallis chi-squared = 41.97, df = 9, p < 0.001) than the tasks create new vector, data aggregation, data imputation and merge data (Figure 7). Again, data type transformation was only used at the beginning of the workflow to transform columns mixing numbers and text to numbers.\n\nLetters refer to: a=create new factor, b=create new vector, c=data aggregation, d=data extraction, e=data imputation, f=data modeling, g=data type transformation, h=merge data, i=modify a vector, j=sort data. The small dots are the relative complexities, the diamonds the means. The whiskers are 25% quantile - 1.5 IQR and 75% quantile + 1.5 * IQR, big black circles are outliers. Signific.: * = 0.05, ** = 0.001.\n\nData usage in relation to data sources was higher in smaller data sources. “Wide” data sources, those consisting of many columns, contributed less to the analysis than “smaller” data sources with fewer columns. While on average 37.4% of the columns in the data sources were used, a linear regression showed that the number of columns not used increased with the total number of columns available per data source (Figure 8).\n\nThe gray shaded area represents the standard error. Linear model with total columns as predictor for unused columns: R-squared: 0.925, F-statistic: 74.02 on 1 and 6 DF, p-value: 0.0001.\n\nAt the same time, data usage in relation to the workflow was similar for all data sources. Data column usage within the workflow ranged between a minimum of 1 and a maximum of 16 with an overall mean of 6.38 (± 4.25 SD). Although usage was different between datasets (Kruskal-Wallis, chi-squared = 18.05, df = 7, p-value = 0.012), a post hoc group wise comparison could not identify the differences (Wilcoxon test). The data column quality, the amount of processing steps needed to transform data for the analysis (see above), was also similar for all data sources. It ranged between a minimum of 1 and a maximum of 10 with an overall mean of 3.54 (± 2.44 SD). There were no differences in the data column quality between data sources (Kruskal-Wallis, chi-squared = 10.9, df = 7, p-value = 0.14).\n\n\nDiscussion\n\nWe showed that workflow complexity and data usage of a typical analysis in BEF can be quantified using relatively simple qualitative and quantitative measures based on commands, code lines, and variable numbers. It is the data aggregation, merging, and subsetting part at the beginning that complicates workflows. In our case, workflow complexity decreased exponentially in the course of the analysis (Figure 4). Similarly,10 found that the data transformation, merging and aggregation steps at the beginning of an analysis complicate workflows. Thus, simplifying data processing steps would greatly increase workflow simplicity. Here we argue that data simplicity could be fostered by providing feedback to data providers on the usage and quality values of the columns in their datasets14.\n\nIn our workflow, “wide” datasets consisting of many columns, contributed less to the analysis than smaller datasets with fewer columns (Figure 8). The more data columns a dataset has, the more difficult it is to understand what the dataset is about. The more data columns it has, the more difficult it is to describe it. The high number of columns in datasets resulting from fieldwork in ecology is a result of the effort to provide comprehensive information in one file only, often including different experimental designs and methodologies. These datasets result from copying field notes that are related to the same research objects, but combine information from different experiments. For example, a field campaign on estimating the amount of woody debris on a study site might count the number and size of branches found. At the same time, as one is already in the field, other branches might be used to find general rules for branch allometries. Thus, the same sheet of paper will be used for two different purposes. While this approach is efficient in terms of time and field work effort, it leads to highly complicated datasets. Separating the datasets into two, one for the dead matter, the other for branch allometries would decrease the number of columns of a dataset and increase the value of the dataset for the analysis of carbon budgets.\n\nCombining data from different sources for meta-analyses could especially benefit from a more atomic way of storing data. Atomic means data particles (e.g. columns) stored separately, described via metadata and linked to ontological concepts. But in ecology the linking of data is rarely performed due to the high heterogeneity of data and concepts. With emerging technologies and a broader acceptance of metadata and ontological frameworks in ecology, datasets could be created automatically using logical constraints built from available atomic data particles. So, a query could return horizontal and vertically subsetted data products (facets) that, in the best case scenario, represent a 100% match directly usable in a meta-analysis20.\n\nProviding feedback to data providers about the complexity of their data may thus be an important step in leveraging the readability of scientific workflows and supporting the reproducibility of data-driven science. This is especially true for “dark” data, the small and complex datasets in the long tail of big data4. We are presently witnessing a growing concern over the loss of data21, which is mostly due to the illegibility of datasets due to missing metadata and the lack of adherence to standard formats. Researchers still do not have training in data management. This concern in losing complex data has led to the invention of tools like DataUP that help to annotate data within Excel, or BEFdata to import Excel files, since this spreadsheet software is mainly used for data storage by researchers. At the same time, opportunities are emerging to publish datasets (Ecological Archives is only one alternative, there are also data journals http://www.hindawi.com/dpis/ecology/) and to provide measures of impact for data22.\n\nProviding means for data quality feedback may also be helpful for propagating data ownership, which remains an unsolved problem14 and a major concern in data sharing23,24. We show that in our analysis, all data columns had a similar usage factor in relation to the workflow. Such usage factors could help to quantify data ownership as they allow one to quantify the amount a certain column or dataset has contributed to derive the results of an analysis.\n\nSince we used the Kepler workflow software to execute R scripts, we made use of Kepler’s interface components. Our text-based approach of quantifying complexity will thus be useful mainly in the context of workflows that work with custom scripts. However, the Kepler workflows are stored as XML files and our approach could thus be generalized to other components in Kepler, or other workflow systems working with XML as an exchange format. Even if workflow programs do not store their workflows in human readable form, their source code could be analysed using similar text-based measures. Providing complexity measures at the level of workflow components might help in re-using and adapting workflows.\n\nTo date, the workflow platform “MyExperiment” is used by 7500 members and presents 2500 workflows for reuse and adaption, however, there is only a rating for the workflow. Offering complexity measures for workflow components may help to identify bottlenecks in existing workflows and help users to adapt components of workflows25, http://www.myexperiment.org/workflows?query=ecology).\n\nA further step in finding and adapting workflows would be the possibility to identify useful workflow components. Here we show that we could identify workflow tasks using text mining (Figure 6). In our case, we could identify one task of very low complexity (data type transformation). This task was very simple and constituted the second axis of our NMDS (Figure 6). Components of this task mostly convert text vectors into numeric vectors. Having text vectors that could actually be interpreted as numbers stems from a “weakness” of EML, in that it does not allow text in data columns that store numbers. However, it is very common that scientists comment missing values or numbers below or above a measuring uncertainty threshold using text. To store the datasets in EML format forces the data provider to label the whole column as a text column.\n\nAs a consequence of having identified this simple and repetitive task of converting text to numbers, we have now added a feature to the BEFdata platform application that automates the conversion. We now offer two ways of exporting the data as comma separated values (CSV), one using the original data and one duplicating numeric columns that contain text, one column containing only the numbers, the other containing only the text. This is also the procedure suggested by DataUP for dealing with columns mixing text and numbers26. The BEFdata EML export now only offers the data in the latter format, so that numbers are no longer mixed with text. This is an example of how the analysis of a scientific workflow can guide towards useful automation features for data repositories.\n\n\nSummary\n\nSimplicity of data sources is the key to simple workflows, but we currently lack feedback mechanisms for quantifying data simplicity. We show that simple text-based measures could already be helpful in quantifying data and workflow complexity. Providing feedback on data complexity as well as the complexity of workflow components may not only foster simplicity and reuse, but additionally may present a means of propagating data ownership through interdisciplinary synthesis efforts and highlights the importance of the underlying primary research data.\n\n\nData availability\n\nfigshare: Data used to quantify the complexity of the workflow on biodiversity-ecosystem functioning, http://dx.doi.org/10.6084/m9.figshare.100831927", "appendix": "Author contributions\n\n\n\nC.T.P., K.N., S.R., C.W. and H.B. substantially contributed to the work including the conceptualization of the work, the acquisition and analysis of data as well as critical revision of the draft towards the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe data was collected by 7 independent projects of the biodiversity - ecosystem functioning - China (BEF-China) research group funded by the German Research Foundation (DFG, FOR 891).\n\n\nAcknowledgements\n\nThanks to all the data owners from the BEF-China experiment who contributed their data to make this analysis possible. The research data is not all publicly available currently but will be in future. The datasets are linked and the ones publicly available are marked accordingly and can be downloaded using the following links. By dataset these are: Wood density of tree species in the Comparative study plot (CSPs): David Eichenberg, Martin Böhnke, Helge Bruelheide. Tree size in the CSPs in 2008 and 2009: Bernhard Schmid, Martin Baruffol. Biomass of herb layer plants in the CSPs, separated into functional groups (public): Alexandra Erfmeier, Sabine Both. Gravimetric Water Content of the Mineral Soil in the CSPs: Stefan Trogisch, Michael Scherer-Lorenzen. Coarse woody debris (CWD): Collection of data on dead wood with special regard to snow break (public): Goddert von Oheimb, Karin Nadrowski, Christian Wirth. CSP information to be shared with all BEF-China scientists: Helge Bruelheide, Karin Nadrowski. CNS and pH analyses of soils depth, increments of 27 Comparative Study Plots: Peter Kühn, Thomas Scholten, Christian Geißler.\n\n\nReferences\n\nMichener WK, Jones MB: Ecoinformatics: supporting ecology as a data-intensive science. Trends Ecol Evol. 2012; 27(2): 85–93. PubMed Abstract \| Publisher Full Text\n\nAltintas I, Berkley C, Jaeger E, et al.: Kepler: an extensible system for design and execution of scientific workflows. Proceedings. 16th International Conference on Scientific and Statistical Database Management. 2004; 423–424. Publisher Full Text\n\nEwa D, Gurmeet S, Mei-hui S, et al.: Pegasus: a framework for mapping complex scientific workflows onto distributed systems. 2005; 13: 219–237. Reference Source\n\nHeidorn PB: Shedding light on the dark data in the long tail of science. Library Trends. 2008; 57(2): 280–299. Publisher Full Text\n\nGries C, Porter JH: Moving from custom scripts with extensive instructions to a workflow system: use of the Kepler workflow engine in environmental information management. In M B Jones and C Gries, editors, Environmental Information Management Conference 2011. Santa Barbara, CA University of California. 2011; 70–75. Reference Source\n\nEwa D, Gurmeet S, Mei-hui S, et al.: Pegasus: a framework for mapping complex scientific workflows onto distributed systems. 2005; 13: 219–237. Reference Source\n\nOinn T, Greenwood M, Addis M, et al.: Taverna: lessons in creating a workflow environment for the life sciences. Concurrency Computation: Pract Exp. 2006; 18(10): 1067–1100. Publisher Full Text\n\nBowers S, Ludäscher B: Towards Automatic Generation of Semantic Types in Scientific Workflows. Web Information Systems Engineering–WISE 2005 Workshops Proceedings. 2005; 3807. : 207–216. Publisher Full Text\n\nMcCabe TJ: A complexity measure. In Proceedings of the 2nd international conference on Software engineering. ICSE ’76, Los Alamitos, CA USA. IEEE Computer Society Press. 1976; 2(4): 308–320. Publisher Full Text\n\nGarijo D, Alper P, Belhajjame K, et al.: Common motifs in scientific workflows: An empirical analysis. IEEE 8th International Conference on E-Science. 2012; 1–8. Publisher Full Text\n\nGil Y, González-Calero PA, Kim J, et al.: A semantic framework for automatic generation of computational workflows using distributed data and component catalogues. J Experimental Theoretical Artificial Intelligence. 2011; 23(4): 389–467. Publisher Full Text\n\nNadrowski K, Ratcliffe S, Bönisch G, et al.: Harmonizing, annotating and sharing data in biodiversityecosystem functioning research. Methods Ecol Evol. 2013; 4(2): 201–205. Publisher Full Text\n\nParsons MA, Godoy O, LeDrew E, et al.: A conceptual framework for managing very diverse data for complex, interdisciplinary science. J Info Sci. 2011; 37(6): 555–569. Publisher Full Text\n\nIngwersen P, Chavan V: Indicators for the Data Usage Index (DUI): an incentive for publishing primary biodiversity data through global information infrastructure. BMC Bioinformatics. 2011; 12(Suppl 15): S3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBruelheide H: The role of tree and shrub diversity for production, erosion control, element cycling, and species conservation in Chinese subtropical forest ecosystems. 2010. Reference Source\n\nFegraus EH, Andelman S, Jones MB, et al.: Maximizing the value of ecological data with structured metadata: an introduction to ecological metadata language (eml) and principles for metadata creation. Bulletin of the Ecological Society of America. 2005; 86(3): 158–168. Publisher Full Text\n\nR Development Core Team. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria, ISBN 3-90005107-0, 2008. Reference Source\n\nBurnham KP, Anderson DR: Model selection and multimodel inference: a practical information-theoretic approach. Springer. 2002; 172. Reference Source\n\nDixon P: Vegan, a package of r functions for community ecology. J Vegetation Sci. 2003; 14(6): 927–930. Publisher Full Text\n\nLeinfelder B, Bowers S, Jones MB, et al.: Using Semantic Metadata for Discovery and Integration of Heterogeneous Ecological Data. Language. 2011; 92–97.\n\nNelson B: Data sharing: Empty archives. Nature. 2009; 461(7261): 160–163. PubMed Abstract \| Publisher Full Text\n\nPiwowar H: Altmetrics: Value all research products. Nature. 2013; 493(7431): 159–159. PubMed Abstract \| Publisher Full Text\n\nCragin MH, Palmer CL, Carlson JR, et al.: Data sharing, small science and institutional repositories. Philos Trans A Math Phys Eng Sci. 2010; 368(1926): 4023–38. PubMed Abstract \| Publisher Full Text\n\nXiaolei H, Hawkins BA, Fumin L, et al.: Willing or unwilling to share primary biodiversity data: results and implications of an international survey. Conservation Letters. 2012; 5(5): 399–406. Publisher Full Text\n\nDe Roure D, Goble C, Bhagat J, et al.: myexperiment: Defining the social virtual research environment. In eScience, 2008. eScience ’08. IEEE Fourth International Conference on. 2008; 182–189. Publisher Full Text\n\nDataUp. The dataup tool. developed by the california digital library and microsoft research connections with funding from gordon and betty moore foundation. 2013. Reference Source\n\nPfaff CT, Nadrowski K, Ratcliffe S, et al.: Data used to quantify the complexity of the workflow on biodiversity-ecosystem functioning. Figshare. 2014. Data Source" }	[ { "id": "4788", "date": "04 Jun 2014", "name": "Paolo Missier", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title and abstract do indeed summarise the purpose and content of the paper adequately.While the goals of the work are laudable, the framework proposed to go about them is not convincing. I see two main problems, firstly with using a single case study to drive the definition and analysis of data and process complexity, and thus to derive results can hardly have general validity. Secondly, by basing the analysis on some questionable assumptions. In what follows, I try to elaborate on these points.The paper makes a strong case for simplicity of data and components, however that is based on a single sample. This is hardly justified, and at odds with the wealth of quantitative research methods machinery deployed to analyse workflows and data and derive the metrics proposed in the paper.It would be good to clarify whether the paper's focus is on the method -- whereby the case study is just an illustrative example, and without any pretense of drawing general conclusions, or on the actual results, which given the very limited evaluation, are questionable.Regarding assumptions, it is stated \"data complexity could be measured by the complexity of their workflow\". How general is this meant to be? I am not sure a process-independent notion of data complexity is given in the paper, but I believe it should be, to clarify the argument. Here complexity seems to be based on how many different usages (and reuse) the data supports, which is fine perhaps, but only one of many possible criteria. I am also suspicious of process complexity criteria based on lines of code, especially in workflows that are composed of discrete components, often pre-existing and part of libraries. Kepler is idiosyncratic in this, as it assumes most actors are ad hoc programs. More generally, workflow is about coarse-grained composition (eg of third party services), and local coding decisions matter a lot less than in hand-crafted code.LOC is a very crude measure of complexity. Just as old, but perhaps more appropriate, is the notion of \"function points\" whereby you express complexity in terms of functionality realised by a component -- regardless of how much code is required to implement a certain function. LOC alone is also at odds with the idea that languages like R sit on powerful packages, which make for succinct but expressive code. How do you compare R code that implements a whole algorithm in R with one that simply invokes a lib function to achieve the same result?One could also argue, reading on pg 7 (col 2), that you may be measuring personal coding style rather that actual process complexity.Other assumptions along the way seem contrived and overfit the (single) example, for instance \"output ports of a data source in the workflow directly relate to data columns in the data set\". (pg 5,6) In the same section, questionable conclusions follow from this assumption.So overall, I think the quantitative methods used in the paper are interesting, but they are applied to a framework where a number of initial assumptions are questionable, and seem to be driven by one single example. A few specific comments:pg 3 - Complexity: The point is about programs with control structures, but scientific workflows traditionally are dataflows. So does the same notion of complexity apply here? pg 4: I feel there is probably too much detail on the science and its results here, which is not the focus of the paper and can be distracting (and uninteresting unless you know the specific science). pg 5 col 2: Need to explain AIC. pg 7: I found table 2 interesting and generally useful. In contrast, Table 3 is a bit of a mystery to me.", "responses": [ { "c_id": "1060", "date": "03 Nov 2014", "name": "Claas-Thido Pfaff", "role": "Author Response", "response": "Dear Paolo Missier,First of all thank you for your valuable input which gave us the opportunity to sharpen the focus of our paper. Your main argument was that we cannot prove our points because we are using a single case study. At the same time you said we should clarify whether our focus is on the results of the analysis - based on only one use case - or the metrics derived for illustrating complexity. However, our main focus is neither on the specific results of this use case, nor on the metrics. We are writing an opinion paper, and both, the use case and the metrics, are illustrations of our opinion. As you say in your comment, - and we take this as a compliment -, we want to “make a strong case for simplicity of data and workflow components”.Although it is not our intention to use the case study as proof, our paper is accompanied by many statistical analyses and plots. This may fool the reader in believing that we want to present a research article. However, we think that our plots are very useful for other data managers and scientists in illustrating why it is worthwhile to invest energy into simplifying datasets. This is especially the case for files from the long tail of big data, which are handcrafted, and relatively small data sets resulting from fieldwork and not from automated sensors. To be able to illustrate the problem of merging these files - which is our day to day work as hybrids of data managers and researchers - we chose this case study, as it is representative for our work and the work of our fellow data managers we spoke to. We also think that it is highly useful to illustrate our difficulties in data re-use.Reworking our text in response to your questions, we scaled down the method descriptions and put a stronger focus on the opinion parts of the paper. We completely reworked the text in many passages and provide here some examples:For example, in the abstract we changed the sentence (page 1):“We illustrate our points using a typical analysis in BEF research...”to:“To illustrate our points we chose a typical analysis in BEF research...”.At the beginning of the introduction we sharpened the opinion aspect of the paper, instead of the sentence (page 2):“However, there is a lack of papers that discuss workflow components within an analysis including data processing.”we now write :“Here we argue that there is a need for quality measures of workflow components, which include scripts, as well as for the underlying data sources. Failure to reuse workflows and available research data is not only a waste of time, money and effort but also represents a threat to the basic scientific principle of reproducibility. Providing feedback mechanisms on the data and workflow component complexity has the great potential to increase the readability and the reuse of workflows and its components.”and other small changes like:before:“We thus suggest that focusing on simplifying ...”after:“We argue that focusing on simplifying...”We further added a new paragraph to the discussion on our methods. We explain, that we want to illustrate the possibility to use simple text mining techniques in providing immediate feedback to data providers or workflow creators. We also add additional avenues that could be taken to quantify complexity of further workflow components or scriptlets (last paragraph discussion):“We here exemplify how to quantify the complexity as well as the quality and the usage of data in scientific workflows, using simple qualitative and quantitative measures. Our means are not meant to be exhaustive but rather could serve as a starting point for discussion towards the development of more sophisticated complexity feedback mechanisms for data providers and workflows creators. Our example workflow strongly relies on the interface component of Kepler connecting to the R statistical environment for the purpose of data manipulation and analysis. Thus the means we provide to measure complexity and quality are adapted to that specific workflow situation. However, adapting our means to further components that work as interfaces to other programming languages should be straightforward. Further complexity attributes could be the inclusion of the variable types of workflow components or a ratio capturing the enrichment or reduction of data consumed by the component. Providing complexity measures at the level of workflow components might help in adapting workflows towards a better readability and reusability and thus improve their value for reuse. Additionally it can guide the restructuring and simplification of data for a better use in workflows, a better understandability and reuse.”We further agree, that we have provided too little explanation of what we mean by “complexity”. We thus added a paragraph in the introduction, section complexity and identity, to define the aspect of complexity we are concerned about (section complexity and identity, first paragraph):“Here we are interested in workflows that begin with the cleaning, the aggregation, and the imputation of research data. These first steps can make up to 70% of the whole workflow. As data managers and researchers, we want to improve the readability of such workflows whether they are scripts or graphs. Our concept of complexity thus should capture the effort and time needed to understand and reuse such workflows. Regarding the complexity of source code we found similar incentives that provide quality measures. The Code Climate service for example provides code complexity feedback to programmers in many different programming languages (https://codeclimate.com/?v=b). Their complexity measures take the number of lines of code as well as the repetition of identical code lines into account.”Our operationalisation of data complexity is based on this approach to workflow complexity. We explain in the same section (paragraph 2 - 3):“Quantifying data complexity is not as straight forward as workflow component complexity. Datasets used for synthesis in research collaborations often consist of “dark” data, lacking sufficient meta- data for reuse….… Here we argue that data complexity can be quantified by looking at the workflow components needed to aggregate and focus the data for analysis. One of the paradigms of data- driven science is that the analysis should be accompanied by it’s data. We argue that at the same time, data should be accompanied by workflows that offer meaningful aggregation of the data. Data complexity could then be measured by the complexity of their workflows.\"More technically speaking, you asked for a process independent complexity measure and criticised the use of line of codes, asking how we deal with hidden complexity when using whole script packages with only one line of code. However, the overwhelming majority of data merging efforts we see in our work as data managers are script based, and are not meant to be reused in the same way as software programs. For this reason, function points do not make sense for them. In addition, we do not only use lines of code in our complexity measure. We also include the number of packages used, for example.In our paper, in the part on the Example workflow, section Quantifying workflow complexity, we explain:To quantify the complexity of the components we used the number of code lines (loc), the number of R commands (cc) and R packages used (pc), as well as the number of input and output ports (cp) of the components (equation 1). However, we are aware that we only use simple and crude methods to assess complexity. As this is not a research paper, but an illustration for an opinion paper, we do not want to focus on the methods too much. On the other hand, we think that it would be good to develop complexity measures for these type of data merging scripts as well as their components and data sources. For this reason we added a whole new paragraph on our methods to the discussion, as stated above.We agree that our measure of complexity is within one personal coding style only. This has the disadvantage that there is only one person or coding style, but the advantage that the differences between the complexities of components is not confused by different coding styles. In most cases, data merging efforts will be done by one person only. We do not want to generalise for all researchers as to which commands or packages they choose. But from our experience, our coding example is representative for data merging exercises. Independently from coding style, most effort goes into the first data cleaning and aggregation steps, including the effort to understand the different data sets. Whatever means we find to give a feedback on how much effort is needed to reuse this data, it is worthwhile giving it back to the data providers.In the following, we answer to specific comments:Paolo Missier: \"Other assumptions along the way seem contrived and overfit the (single) example, for instance \"output ports of a data source in the workflow directly relate to data columns in the data set\". (pg 5,6) In the same section, questionable conclusions follow from this assumption.\"We agree that this formulation is misleading. Since we use the EML actor of Kepler to import data, the “output ports” are always the data columns. We did not want to imply a causality here. Data columns appear as output ports in the Kepler actor, because this is how the EML actor works. We reformulate this sentence accordingly. Indeed, the paragraph works without even using the whole sentence (see page 3, section: quantify quality and usage of data)Before:“As explained above, output ports of a data source in the workflow directly relate to data columns in the data set. Thus, the number of available ports of a data source is the “width” of a dataset, or the number of data columns. Thus, the usage of a data column in rela- tion to the data source was calculated as the ratio of ports actually used in the workflow to the ports that were not used. This allowed us to relate the number of unused ports to the number of available ports of a data source.\"Now:“For our analysis we only used a subset of the data columns available in each data source. We therefore quantified the “data usage” of a data source as the ratio of data columns used for the analysis to the total number of data columns in that data source.\"Paolo Missier: \"pg 3 - Complexity: The point is about programs with control structures, but scientific workflows traditionally are dataflows. So does the same notion of complexity apply here?\"No, it doesn’t. We now provide a definition of complexity that clarifies that we are interested in the amount of effort and time that has to be invested in data or workflow reuse (see above).Paolo Missier: \"pg 4: I feel there is probably too much detail on the science and its results here, which is not the focus of the paper and can be distracting (and uninteresting unless you know the specific science).\"We have reordered and shortened the paragraphs on our workflow example. However some the information is interesting for the general reader and are required for the overall understanding. For example that the data sources come from independent projects and are archived in a common platform as well as some basics on workflows. But we have shortened the information on the scientific analysis to one paragraph.page 3, section: biodiversity effects on subtropical carbon stocks:“Our example workflow is part of an ongoing study that measures biodiversity effects on subtropical carbon stocks and flows. It is typical for synthesis tasks in collaborative research projects in that it combines eight datasets collected by seven independent research groups collaborating within the BEF-China research platform (www.bef-china.de). Data is archived, harmonized, and exchanged using the BEFdata web application (citation!!!). Data is exported in EML format and as such imported into the Kepler Workflow system.The data describes carbon pools from soil, litter, […] from the years 2008 and 2009 on the observational plots of the BEF-China research platform spanning a gradient from 22 to 116 years of plot age and 15 to 35 tree species. Our example workflow merges the data and terminates in a linear model relating biomass pools to plot age and plot diversity. It shows that carbon pools increase with stand age, however, in plots with high species richness this increase was less steep (p-values).”Paolo Missier: \"pg 5 col 2: Need to explain AIC.\"AIC is explained and cited in the methods part (Akaikes Information criterion). page 3, right column, section: quantify component identity:“For this we used linear models which have been compared using the Akaike Information Criteria (AIC) to select for the most parsimonious model.”Paolo Missier: \"pg 7: I found table 2 interesting and generally useful. In contrast, Table 3 is a bit of a mystery to me.\"Table 1, 3 and Figure 6 are different perspectives on the same topic. Figure 6 shows the ordination result using multidimensional scaling (NMDS) of workflow component characteristics. The NMDS results in 2 axes that span the highest variation of components in the characteristics space. Thus NMDS1, the first axis, spans the highest variation between components. The component identities in Table 1 as well as the component characteristics in Table 3 were later compared to the axes scores of the NMDS axes. These are the measures given in Table 2. We have changed the text in the captions to point out the relatedness of the figure and the tables. We additionally included and example in how to interpret measures in Table 2 when comparing them with Figure 6.Table 1:“Workflow component identities defined a priori and their relation to the data oriented motifs identified by (10). Figure 6 plots the a priori defined identities of the workflow components to the characteristics we measured from each component a posteriori. Characteristics include lines of codes or specific commands (Table 3). ”Table 2:“Characteristics of workflow components used to assess variation between components by means of non metric multidimensional scaling (NMDS). Characteristics include lines of code, use of packages, as well as specific commands (see text for further detail). Figure 6 plots the two axes of the NMDS. r2, Pr(>r) and sig. describe R square, Probability, and significance level of a correlation with the characteristic as dependent and the NMDS scores of both axes (NMDS1, NMDS2) as independent variables. For example, “count of code lines” separates workflow components in the NMDS plot, so that components with more code lines are plotted in the upper left quadrant of the plot in Figure 6. Signif. codes…”Figure 6:“Workflow components (points) in reduced component characteristics space (Table 3). We used non metric multidimensional scaling (NMDS, see text for further detail) to reduce the parameter space to two axes. Table 3 lists the regression results of the axes scores on the component characteristics, which are plotted in smaller text here. Table 2 lists the a priori tasks, which are plotted in large labels here. Points are jittered by a factor of 0.2 horizontally and vertically to handle over plotting.”" } ] } ]	1	https://f1000research.com/articles/3-110
https://f1000research.com/articles/3-281/v1	14 Nov 14	{ "type": "Research Article", "title": "Effect of microalgae application on soil algal species diversity, cation exchange capacity and organic matter after herbicide treatments", "authors": [ "Karin L. Hastings", "Lauren E. Smith", "Michael L Lindsey", "Luke C. Blotsky", "Gloria R. Downing", "Derex Q. Zellars", "Jason K. Downing", "Maria Corena-McLeod", "Karin L. Hastings", "Lauren E. Smith", "Michael L Lindsey", "Luke C. Blotsky", "Gloria R. Downing", "Derex Q. Zellars", "Jason K. Downing" ], "abstract": "Soil microalgae live on small communities that change structure depending on many factors. Some of these factors include soil pH, agricultural practices as well as pesticide and herbicide treatments. The size and activity of these soil microbial communities is an indicator of health, quality and fertility necessary for sustainable agriculture. Methods: A commercial proprietary suspension of microalgae GOgreen® was applied at different concentrations through a center pivot irrigation system to a crop of cultivated corn (Zea mays) during six months. Experimental plots of 0.5 acres each were planted in four rows. Corn (34,000 plants/acre) was planted in May and harvested in November allowing each plot to be studied for 6 months. In addition, one plot was planted for a second year to study the effects of consecutive planting and harvesting on algae populations in the soil. Soil samples were collected at a depth of 6 inches. Microalgae species identification was based on morphological criteria. Soil analysis included organic matter content (OM), pH and cation exchange capacity (CEC). Results: Treatment with GOgreen® has significant and measurable positive effects on soil OM content, CEC, pH and microalgae species diversity. Conclusions: It was demonstrated through this study that GOgreen® increased diatom numbers and microalgae species diversity showing a restorative effect on soil quality after herbicide treatment in heavily farmed soil. Additionally, GOgreen® lowers the pH in soils with a pH higher than 7.0 emerging as an economical alternative that is safe for humans and the environment.", "keywords": [ "Microalgae", "Cation Exchange Capacity", "Organic Matter", "Herbicides", "Corn", "Crops", "Fertilizer", "Green", "GOgreen", "pH", "restoration", "species diversity", "microalgae", "soil algae", "soil amendments", "soil biodiversity." ], "content": "Introduction\n\nThe size and activity of soil microbial communities is an indicator of soil health, quality and fertility necessary for sustainable agriculture (Doran & Parkin, 1994; Kennedy & Papendick, 1995; Sparling, 1997; Warkentin, 1995). These communities have been classified into eubacteria, cyanobacteria, actinomycetes, archaebacteria, fungi, microalgae, protozoa, viruses, and some nematodes (Paul & Clark, 1989; Roper & Gupta, 1995; Sims, 1990). Out of these groups, microalgae perform several important functions for agro-ecosystems and can also function as a bio-indicator for soil quality.\n\nFour different types of algae are recognized in soil: green (chlorophyta), blue-green (cyanobacteria), yellow-green (xanthophyta), and diatoms (bacillariophyta) (Paul & Clark, 1989). Soil algae are photoautotrophs. These species do not depend on the organic matter (carbon content) of the soil and play a role as primary colonizers. They produce large amounts of secreted polysaccharides that promote soil aggregation at the surface and they are capable of nitrogen fixation (Goyal, 1997; Zenova et al., 1995). Soil habitats are the most important non-aqueous ecosystems for microalgae (Zenova et al., 1995) where these organisms contribute to soil formation and stability (Metting, 1981). In addition, microalgae contribute to energy and matter flux (Kuzyakhmetov, 1998). Green and blue-green microalgae populations in upper topsoil can perform valuable services for soil ecosystems (Metting, 1981; Starks et al., 1981) and agriculture (Ruble & Davis, 1988). One of the major benefits of microalgae is the generation of organic matter from inorganic substances (Alexander, 1977). In addition to providing a food source for other microorganisms, nematodes, and invertebrates, microalgae produce biologically active compounds such as enzymes and ions that can affect other components of soil communities, including plants (Metting, 1981; Zenova et al., 1995).\n\nIt has been demonstrated that Gram-negative photosynthetic bacteria play a key role in ecological and plant community succession (Metting, 1981). Primary succession begins in new habitats and it is not influenced by pre-existing communities. Secondary succession follows the disruption of pre-existing communities through external agents such as harvesting, drought or fire. There are few reports on the role that microalgae play in ecological succession. The use of microalgae as fertilizer has recently been suggested (Gaydon et al., 2012). The first objective of this study was to determine the effects of the application of a commercial proprietary suspension of microalgae (Chlorella sp. 1×10e3 cells per mL, Nannochloris sp. 1×10e3 cells per mL, Scenedesmus sp. 1×10e3 cells per mL) formulated to provide nutrients to indigenous soil microorganisms and facilitate microbial density and diversity, as well as to increase soil carbon. The formulation (GOgreen® (Global Organics Group, LLC, Goodyear, AZ) was applied through a center pivot irrigation system in a crop of cultivated corn (Zea mays) on soil algae species diversity after herbicide application and after harvest.\n\nAutogenic and allogenic successions within a particular soil ecosystem are of significant importance to soil communities. In contrast to allogenic succession, caused by abiotic factors such as temperature, light or moisture, autogenic succession is observed when changes in the soil are caused by naturally occurring organisms and plants (Martin & Hine, 2008). These changes include accumulation of organic matter as well as changes in soil nutrient composition or soil pH. Percent organic matter (%OM) is a direct measurement of the amount of organic material (animal and plant residues) in the soil (Bot & Benites, 2005). Organic matter acts as a reservoir for essential nutrients such as nitrogen to be used by the plant or by microbial communities and it is closely related to the Cation Exchange Capacity or CEC of the soil. CEC is directly related to the total amount of cations that a particular soil can hold and in turn it is directly related to the ability of the soil to hold plant nutrients. As OM contributes to cation exchange, the larger the OM content of the soil, the larger the CEC (CUCE, 2007). The secondary objective of this study was to determine the effects of GOgreen® on %OM and CEC in experimental plots with different pH and to compare them with those obtained without GOgreen® treatment.\n\n\nMaterials and methods\n\nExperimental plots of approximately 0.5 acres each were planted in four rows. In order to study the effects of consecutive planting and harvesting on algae populations in the soil, one plot (plot 3) was planted in May 2010 and harvested in October 2010. Treatment for plot 3 is shown in Table 1.\n\nPlot size: 4 rows per variety. Planting population: 35,500. NPK: Nitrogen, Phosphorus and Potassium content.\n\nPlots were designated by numbers based on the application rate of GOgreen®. Plot 1 (10 oz/acre), plot 2 (8 oz/acre), plot 3 (10 oz/acre) and plot 4 (20 oz/acre). A control plot (plot 5 no GOgreen®) was also included. Corn (34,000 plants per acre) was planted in May 2011 and harvested in November 2011 allowing each plot to be studied for 6 months. Treatment for all plots is described in detail in Table 2.\n\nPlot size: 4 rows per variety. All plots were divided into high (8.3) and low pH (6.8). Planting population: 34,000. (N/A = not applicable; second treatment in consecutive years).\n\nSoil samples representative from each of the experimental and control plots were taken at a depth of 6 inches immediately after herbicide application (as described in Table 1) following established protocols at the Irrigation Research Foundation (IRF) in Yuma, CO, USA. These samples were labeled pre-treatment. Analysis included organic matter (OM) content, cation exchange capacity (CEC) and pH. Analysis was performed by Midwest Laboratories, Omaha, NE. Details of protocols are available at https://www.midwestlabs.com/wp-content/uploads/2012/09/139_soil_analysis_methods.pdf.\n\nEach plot received all treatments listed in Table 1 and Table 2 except for the controls. Controls were not treated with GOgreen®. Prior to harvest, soil samples were taken from each of the plots (and control). These samples were labeled post-GOgreen®. Samples were also taken from each of the plots and controls two months after harvesting. These samples were labeled post-GOgreen® two months. Each plot crossed a region of high soil pH (8.3) and neutral soil pH (6.8) and soil samples were taken individually from these regions. Treatments are described in Table 2. After being in use in 2010, plot 3 was again planted in May 2011 and harvested in November 2011 allowing for a year of consecutive corn harvesting and data recording on this plot.\n\nDescription of herbicide treatments was relevant to our study because herbicides have been documented to have a detrimental effect on soil algae populations (Kuzyakhmetov, 1998; Zancan et al., 2006). GOgreen® was delivered at the manufacture’s recommended application rate through center pivot irrigation before the V7 stage of corn plant growth. Soil samples were taken at V5 stage of plant growth as well as 4, 6, 7, and 8 weeks after emergence. A final sample was taken at harvest (black layer).\n\nTwo-tailed paired difference t-test was used to compare groups pre and post treatment to determine if GOgreen® had an effect on the tested soil parameters. Results of Statistical analysis are shown in Table 4. Statistical analysis was performed using GraphPad Prism software (GraphPad, San Diego, CA, USA).\n\nMedia and solutions were prepared using reagents from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. HEPES buffer (50 mM, pH 7.8) was used to prepare Vitamin B12, Biotin and Thiamine solutions. In order to culture algae for identification purposes, 1 gram of soil from each plot, including high and low pH regions as separate samples, was added to 10 mL of selective media containing BG-11, Bolds 3N and DM and plated using approximately 200 μl in Agar plates with the same composition. Plates were prepared using a spread plate method. The media was selective for photosynthetic organisms.\n\n3N Modified Bold’s Basal Media (Bold, 1949) was used for enrichment of green, red, and brown algae. Soil water was prepared using an adaptation of E.G. Pringsheim's biphasic soil-water medium (Pringsheim, 1946). Diatom Medium (DM) was used to culture and identify diatoms (Beakes et al., 1988), Proteose medium (PM) was used for yellow-green algae. PM was made by adding proteose peptone to Bristol Medium at a final concentration of 1 g/L. Bristol medium was prepared according to Bold, (1949). To culture cyanobacteria, BG-11 was used as previously described (Stanier et al., 1971). Cultures were grown at 16:8 under full spectrum grow lights for 4–6 weeks at 22°C. Algae layers (1 mL) were collected using a Pasteur pipette and plates of selective medium were inoculated. Colonies were counted and identified through light microscopy using a Labomed LX500 microscope (Labomed, Hicksville, NY) six days dates post-inoculation.\n\n\nResults\n\nResults from soil analysis pre-treatment and after GOgreen® treatment at harvest and 2 months post-harvest are shown in Table 3. Plot 2 values are shown prior to plot 1 values as the dose applied in plot 2 was 8 oz./acre vs. 10 oz./acre applied in plot 1. This order facilitated comparison between a one time application of GOgreen® (plot 1) vs. consecutive GOgreen® application (plot 3). Percentages of OM were very low for all plots including the control plot. This is typically observed in soils in the Southwest (USA) (Hargrove & Luxmore, 1988). Differences in OM and CEC pre- and post-GOgreen® treatment for each individual plot are shown in Figure 1A and B respectively.\n\nAll units in parts per million (ppm) unless otherwise noted. indicates pre- GOgreen® treatment the previous year. Abbreviations: OM (%) = Percent Organic Matter, CEC= Cation Exchange Capacity. L = pH < 8.0, H = pH > 8.0 post = after GOgreen® treatment. Post-2M = 2 months after harvest.\n\nGiven soil heterogeneity plots were grouped in pre and post treatment groups. Paired Difference t-test analysis was chosen given the small number of samples to analyze. Stedv (Standard Deviation of the Population), df = degrees of freedom.\n\nA. In soil with pH < 8.0 (red) a lower OM content when compared to the control was observed. These numbers increased 2 months after harvest as shown to the right. B. Soils with pH > 8.0 (green) showed the opposite trend. These changes indicate that treatment with GOgreen® result in changes in OM content in the soil that vary according to pH.\n\nOur results indicate that GOgreen® has a significant effect in the OM content of soil at both pH values tested. Although lower OM values were observed prior to harvest in soil treated with GOgreen® with pH < 8.0 when compared with the controls (Figure 1A (left)), an increase in OM was observed two months after harvest (Figure 1A (right)). In soil with pH > 8.0, GOgreen® OM content values were higher prior to harvest when compared to those obtained at pH < 8.0. In the first group, treatment with GOgreen® at the recommended manufacturer dose (Table 2) appeared to have a positive effect on CEC. Although GOgreen® initially increased the OM content of the soil prior to harvest (Figure 1B (left)) this effect was not sustained two months after harvest (Figure 1B (right)) in this particular soil and/or crop type. It is worth noting that although the control at pH < 8.0 did not show any changes in OM over time, the control at pH > 8.0 showed a significant decrease in OM (shown as a * in Figure 1B) indicating that other factors independent of GOgreen® application might have played a role in OM content measurements at high pH values.\n\nIn terms of CEC, significant differences were observed in CEC two months after harvest when compared to samples collected prior to harvest (Figure 2A and B). It was observed that prior to harvest GOgreen® treatment at the recommended 10 oz./acre increased the CEC of the soil in the treated plots at pH < 8.0. The highest CEC obtained prior to harvest was observed at 20 oz./acre in the soil samples with pH < 8.0. Two months after harvest, the CEC values declined for all plots in this group including the control and the values were uniform. The average CEC for all plots at harvest was 10.6 meq/100g for the controls at this pH value and the average for all plots was 10.68 meq/100g indicating an increase of 0.08 units reflected in a greater ability of the soil to bind and retain ions and nutrients (Table 3). There were no increases observed in CEC in the plots treated with GOgreen® at pH > 8.0 prior to harvest. Although the CEC values for the plots in the pH > 8.0 group were significantly higher (by about 7 meq1/100 g) when compared to the plots in the pH < 8.0 group, two months after harvest, CEC values were also lower than those observed prior to harvest consistent with the CEC values observed for the pH < 8.0 group.\n\nA. Areas with pH < 8.0 (red) show a lower CEC 2 months after treatment, B. Areas with pH > 8.0 (green) show a similar trend with a significantly lower CEC 2 months after harvest.\n\nThe effect of GOgreen® was studied in plots before and after application. Total algae counts were grouped as percentages of blue-green and green algae as well as diatoms. The results are shown in Figure 3 (and Dataset 1).\n\nEffects of pH in total counts of groups of species. A. Total counts all plots pre-treatment. B. Control pre-treatment. C. Total counts all plots pH < 8.0. D. Control pH < 8.0. E. Total counts all plots pH > 8.0. F. Control pH > 8.0. G. Total counts all plots 2 months after harvest. H. Control 2 months after harvest. Please see Dataset 1 for the raw data.\n\nOur results indicate that prior to GOgreen® treatment, the total microalgae population in the soil of the test plots was composed of 60% blue-green algae, 25% green algae and 15% diatoms (Figure 3A). The ratio of green to blue-algae was 0.42. In the control plot, the ratio of green to blue-green algae pre-treatment was approximately 0.45 (Figure 3B) indicating uniformity in the distribution of microalgae by plots before treatment.\n\nIn soils with pH < 8.0, green algae were more abundant, which also correlated with the control at this pH (Figures 3C and D). The amount of diatoms was higher in the treated plots than in the controls. In soils with pH > 8.0, green algae counts were lower than those of blue algae (Figures 3E and F).\n\nThe results obtained two months after harvest indicate the percentages of blue-green algae and green algae were similar after GOgreen® treatment (Figure 3G). In contrast, the controls plots showed a 50% reduction in the blue-green algae counts when compared to the number of green algae (Figure 3H). Although diatoms also appeared in the controls two months after harvest, their counts were significantly higher in the treated plots as shown in Figure 3G.\n\nThe comparison between the pH < 8.0 and the pH > 8.0 controls indicates that there were small differences between their values (1.53 vs. 1.11 respectively). However, the difference between green and blue-green algae in the treated plots was significant (4.69 vs 1.87) indicating differences in microalgae diversity based on pH. In both cases, the ratio in terms of percentages was higher than the ratio of the controls, indicating that treatment does have an impact on microalgae populations. Over time, all treated plots showed higher counts of green algae than blue-algae. When compared to untreated plots, those numbers showed a significant increase as the controls pre-treatment indicated higher numbers of blue-green algae in the soil. Furthermore, diatom concentrations increased in all treated plots. The diatom counts were much higher in soil with pH > 8.0.\n\nThe results of this study indicate differences between soil pH in the plots with pH < 8.0 treated with GOgreen® two months after harvest (Figure 4A). Although the control plot did not show a substantial change in pH (initial pH was 6.9 and pH after harvest was 7.0), the treated plots did show substantial changes over time. A trend based on rates of application was not obvious.\n\nA. Comparison of pH values observed at harvest and two months post-harvest in soils with pH < 8.0. Blue: pH values for plots at harvest. Red: pH values for plots 2 months after harvest. B. Comparison of pH values observed at harvest and two months post-harvest in soils with pH < 8.0. Blue: pH values at harvest. Green: values 2 months after harvest.\n\nIt is clear from Figure 4A that in plots with soil pH > 7.0 at the end of harvest, the pH of the soil reached values closer to neutral two months later. In soils with pH > 8.0 the trend was not as noticeable. However, pH decreased to values closer to 7.0 in plots 1 and 2, as well as the controls.\n\nAn observation worth noting is the difference in pH in plots 3 and plots 4 at pH > 8.0 when compared to the other plots as seen in Figure 4B. At this pH, treatment with GOgreen® the previous year (plot 3) and with twice the recommended application rate (plot 4) resulted in a pH increase of 0.2 units two months after harvest when compared to the other plots. This result contradicts the observations in which pH decreased in the soil two months after harvest in plots 1 and 2.\n\n\nDiscussion\n\nA conventional approach was implemented to evaluate microalgae diversity in soil samples collected at a depth of 6 inches after herbicide and fertilizer treatment. Soil was treated with GOgreen® in an effort to restore and improve microalgae diversity and soil properties. This method included isolation, culture and species identification based on morphological criteria. This method has been successfully used by other investigators (Kostikov et al., 2001).\n\nThe results presented in this manuscript demonstrate that treatment with GOgreen® has significant and measurable effects on soil OM content, CEC, pH and microalgae species diversity suggesting positive effects of this formulation on soil conditions, making them more suitable to sustain crops. Although studies in soil are affected by a variety of biotic and abiotic factors, the effects of GOgreen® are evident in terms of increases in microalgae species diversity. Abiotic factors considered in this study included light intensity, temperature and humidity. Control of external biotic and abiotic factors in the soil is extremely difficult and therefore the best approximation that could be done in a study of this nature is to compare soils that have been treated under the same conditions with simultaneous sampling.\n\nTemperature changes in the environment during the time of the study were considered since it has been reported that blue-green algae are photo inhibited by high light intensities at low temperatures. Temperature can be considered as the most important limiting factor in outdoor cultivation during the winter (Malakar & Kalita, 2012). Blue-green algae growth is enhanced by increasing light density up to the point of light saturation, at which point photosynthetic activity reaches its maximum (Abu et al., 2007; Pandey & Tiwari, 2010). At high light densities, photosynthetic capacity decreases and blue-green algae growth is inhibited. As soil samples were taken from all plots during the cold months of November and February, it is possible to hypothesize that the low counts of blue-green algae found in the treatment plots and the controls after GOgreen® treatment are directly related to changes in ambient temperature and light intensity between soil sampling times. This could be a possible explanation for the differences encountered in the parameters measured between pre and post GOgreen® applications. However, as all plots (treated and not treated) were exposed to the same environmental conditions, it is highly unlikely that the differences found in the treated plots versus the control plot were due to cold temperatures or intense light. The difference must be related to different soil characteristics as a result of the treatment.\n\nDespite abundant studies on soil algae (Johansen, 1993; Metting, 1981; Lukešová, 1993; Lukešová, 2001; Lukešová & Hoffmann, 1996; Neustupa, 2001; Starks et al., 1981; Sukala & Davis, 1994; Tsujimura et al., 2000), it is still difficult to correlate species diversity and their influence on ecosystem functions. Therefore, this manuscript does not attempt to correlate species diversity with metabolic, ion or gas exchanges. Instead, the focus lies on algae species diversity after use of an organic formulation such as GOgreen® and to correlate these findings with indicators of soil quality such as OM and CEC.\n\nThe primary objective of these studies was to determine the effects of GOgreen® on algae species diversity after herbicide application. The results of this study support the findings that Zancan and Zuzyamkhmetov encountered in corn fields subjected to lengthy periods of intense fertilization. Their results indicate a reduction in species diversity and a suppression of blue-green algae development in fields treated with fertilizers and herbicides (Kuzyakhmetov, 1998; Zancan et al., 2006). The control shown in Figure 3B, indicates low species diversity (diatoms are absent) encountered in the soil prior to GOgreen® treatment but after fertilizer and herbicide treatment. The data shown in this manuscript confirms the profound effect of agricultural practices including herbicide and pesticide application on the structure of soil algal communities.\n\nHerbicides have also previously been correlated to changes in microalgae populations in aquatic ecosystems (Bérard & Benninghoff, 2001; Bérard et al., 1999) and decreased density of algal assemblages in plots. Lenacil and Pyrazon for example, have been shown to diminish species diversity and decrease microalgae counts in soil (Zurek, 1981). Herbicides and pesticides influence the range of genera and the number of algal cells, blue-green in particular, present at any given time in vitro and in vivo (McCann & Cullimore,1979; Megharaj et al., 1999; Metting & Rayburn, 1979; Mostafa & Helling, 2002).\n\nOne of the main groups considered the diatoms a valuable tool to assess biological conditions in wetlands (Doherty et al. 2000; Stevenson, 2001). The response of diatoms to changes in surrounding land and water column characteristics has been documented previously and many diatom taxa have been identified from a range of sites throughout the world (Stevenson, 2001). Diatoms appear to have a consistent tolerance of a wide range of environmental parameters, such as light, moisture, pH, salinity, oxygen and inorganic and organic nutrients (van Dam et al., 1994). Responses to pH (Pan & Stevenson, 1996) and heavy metal loading (Charles et al., 1996) have also been used to predict environmental pollution. The US Environmental Protection Agency (USEPA) (2002) reported that diatoms are one of the most commonly used microorganisms in observations from aquatic ecosystems for assessing biological, physical, and chemical conditions. Through this study, it can be concluded that GOgreen® increased diatom numbers and species diversity in the treated plots compared to the controls indicating that GOgreen® has a restorative effect on soil quality after herbicide treatment in heavily farmed soil. Additionally, these results indicate that the observations from aquatic ecosystems can be extrapolated to terrestrial environments.\n\nIn terms of specific differences in species diversity related to green and blue-green algae, a 17% increase was observed in the total counts of green microalgae in the GOgreen® treated plots at pH < 8.0 when compared to the control, while a 22% decrease was observed in blue-green algae in the same plots. Contrary to these results, a 22% decrease in green microalgae was observed in the GOgreen® treated plots at pH > 8.0 when compared to the controls, while a 30% decrease was observed in blue-green algae counts when compared to the control at this high pH value. These results indicate that the effects of GOgreen® application are different and highly dependent on soil pH.\n\nThe pH of all plots tested varied between 6.9 to 8.4, therefore many of the microalgal classes were represented. Blue-green algae are unable to survive in acidic conditions (Brock, 1973), but green algae are able to survive in soils with pH < 7.0 (Lukešová & Hoffmann, 1995). Neutral conditions support the growth of algal communities representing all major taxonomic groups (Lukešová, 2001; Metting, 1981). The percentage of blue-green algae observed in the control plot at pH < 8.0 was 38% (Figure 3D) versus 45% in the control plot at pH > 8.0 (Figure 3F), which supports the findings by the above mentioned investigators. In contrast, the values for green algae in the two controls were similar (58% and 50%). These results indicate increased microalgal species diversity in the GOgreen® treated plots versus the controls within the pH range tested.\n\nAn increase in diatom numbers and diversity was observed at all pH values when treatment plots were compared to controls not treated with GOgreen®. The results of treatment with GOgreen® were different between soil with pH < 8.0 and soil with pH > 8.0. At all pH values, an increase in diatom concentration was observed in the GOgreen® treated plots when compared to the controls, but this effect was higher in soils with pH > 8.0, resulting in a 57% of total diversity composed of diatoms (Figure 3E) versus 9% of diatoms at pH < 8.0 (Figure 3C). The main species found in the study were: Pennate diatom, Caposira, Raphid diatom, Geminella, Navicula diatom, Centric diatom, Tessillaria, Pinnularia.\n\nThe second objective of these studies was to determine the effect of GOgreen® on OM and CEC. Both OM and CEC depend on soil pH. Optimum pH for corn ranges between 5.5–7.0 (Havlin et al., 1999). Our results suggest that soils starting at pH < 8.0 are most likely to fall within the optimal pH range for corn after GOgreen® treatment.\n\nSoil OM serves multiple functions including nutrient storage and soil aggregation. Soils with high CECs are able to bind more monovalent and divalent cations through available sites in clay and OM particles. A soil with a high CEC also has an increased buffering capacity indicating that this soil is able to resist fluctuations in pH.\n\nSoils with a high clay content and/or OM will typically have higher CEC and buffering capacity than silty or sandy soils, as organic materials provide additional binding sites for cations (CUCE, 2007). In this study, high OM correlated with high CEC values of the plots before GOgreen® application. At harvest, CEC was higher in soil with pH > 8.0, which is consistent with the presence of negative charges in the absence of acidic values that in turn have the ability to bind cations. Two months after harvest, values for CEC were lower at both pH ranges as it is expected after harvest due to soil depletion. These changes also correlated with pH values two months after harvest as the pH lowered for both types of soil. The most interesting observation was the fact that the CEC was around 8.0 at both pH values two months after harvest indicating a stabilization of CEC in the soil regardless of pH. Although this value is lower than those encountered at harvest, a low CEC value indicates that fewer cations such as K+, Ca2+, Mg2+, to name a few, will be bound to soil particles and more available for nutrient uptake by the plant. Nitrogen will also be more available and less lime will be necessary to correct pH fluctuations. In soils with pH around 8.0 such as those found in the Western U.S., large amounts of naturally-occurring lime are typically responsible for the increased pH. This “free lime” buffers pH in the alkaline range making it very difficult to change soil pH. Calcium carbonate (CaCO3) is commonly found in these soils. For these particular soils, larger quantities of amendments are needed to lower the pH converting pH modification in alternatives that are not cost effective. Addition of organic matter is typically used to lower pH but not all sources of organic matter are effective or safe for human consumption. GOgreen® lowers the pH in soils with a pH higher than 7.0 emerging as an economical alternative that is safe for humans and the environment.\n\nIn soil, the presence of living organisms has proven critical to OM formation. Soils with OM values around 1.0% are typically found in the desert (CUCE, 2007). Soil OM – the product of on-site biological decomposition – affects the chemical and physical properties of the soil and its overall health. Its composition and breakdown rate affect the soil structure and porosity, the water infiltration rate and moisture holding capacity of soils, the diversity and biological activity of soil organisms, and plant nutrient availability (Bot & Benites, 2005). Where the rate of OM addition is less than the rate of its decomposition, soil OM declines. Conversely, where the rate of addition is higher than the rate of decomposition, soil OM increases. The observations presented in this study regarding the differences in OM at the pH values tested indicate that the rate of OM addition after GOgreen® application is higher than the rate of decomposition in soils with pH < 8.0. The opposite is true for soils with pH > 8.0. However, further studies are needed to test this hypothesis. As changes in OM are lower than 1–2% per year of the total OM in the soil, the effects of GOgreen® algae application will only become significant after several years. For this purpose, this study has been extended in order to monitor the plots described during subsequent years of continuous harvest usage.\n\nAs land available for cultivation becomes scarce due to increasing populations, the need for improved soil quality is now recognized at a global scale (Elliott et al., 1996). Soil communities co-exist in constant interactions with each other (Gerson, 1974; Thirup et al., 2000; Yeates et al., 1993). These interactions are crucial to regulate soil activity and may be disturbed by soil pollution, herbicides, pesticides, fertilizers and management practices (Pankhurst, 1997; Paoletti et al., 1988).\n\nMethods to determine soil quality are varied but scholars have identified microorganisms as potential bio indicators of soil quality (Pipe & Cullimore, 1980; Roper & Opel-Keller, 1997). It has been proposed that approaches to study ecological and eco-toxicological impacts of management and agricultural practices on soil quality should be monitored with the use of multimicrobial bio indicators (Bérard et al., 2005). Soil microalgae depend on soil physical and chemical characteristics and therefore, these organisms have been used for decades as bio indicators to estimate the eco-toxicological impact of agricultural management practices and herbicide application (Fujita & Nakahara, 1999; McCann & Cullimore, 1979; Pipe, 1992). Biodiversity is therefore an indicator of soil quality and a crucial component to evaluate the success of a particular remediation strategy. The use of biodiversity as an indicator is limited by the incomplete knowledge regarding microorganisms present in a particular ecosystem (Paoletti, 1999).\n\nMicroalgae, and diatoms in particular, respond to different ecological gradients and are therefore useful tools for bio monitoring studies in aquatic and terrestrial ecosystems. It has been demonstrated that diatom diversity tends to be higher in biodynamic systems than in conventional systems. Redundancy analysis (RDA) has suggested that diatom community structure differs significantly between organic and conventional systems (Heger et al., 2012).\n\nMicroalgae concentrate in the top few inches of the soil because they need moisture and light to perform photosynthesis. They are located in the first biological layer directly affected by environmental changes, both natural and manmade. Consequently, microalgae composition varies in the soil over several weeks depending on treatment and their numbers are highly influenced by environmental and seasonal factors specific to each region (Hoffmann, 1989; Metting, 1981; Whitton, 2000). These results corroborate previous findings and highlight the importance of CEC, OM and pH variations in increased microalgae species diversity through GOgreen® application.\n\n\nData availability\n\nF1000Research: Dataset 1. Microalgae counts raw data, 10.5256/f1000research.4016.d36846 (Hastings, 2014).", "appendix": "Author contributions\n\n\n\nKH, ML, LB and GD conceived the study. KH and LS designed the experiments. KH, LS, GD, JD, and DZ carried out the research. LB and ML contributed to the design of experiments and provided expertise in rates of application, costs and budgets. MCM prepared the first draft of the manuscript. KH, JD, DZ, ML, MCM and GD contributed to the experimental design and preparation of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was funded by Global Organics, LLC. Goodyear, AZ, USA.\n\n\nAcknowledgements\n\nThe authors would like to thank Irrigation Research Facility (IRF) Yuma, CO and Midwest Labs Omaha, NE for their help with plot and crop treatments and collection.\n\n\nReferences\n\nAbu GO, Ogbonda KH, Aminigo E: Optimization studies of biomass production and protein biosynthesis in a Spirulina sp. isolated from an oil-polluted ﬂamepit in the Niger Delta. Afr J Biotechnol. 2007; 6: 2550–4. Reference Source\n\nAlexander M: Introduction to Soil Microbiology. Wiley, NY. 1977; 467. Reference Source\n\nBeakes G, Canter HM, Jaworski GHM: Zoospores. ultrastructure of Zygorhizidium affluens Canter and Z. planktonicum Canter, two chytrids parasitizing the diatom Asterionella formosa Hassall. Can J Bot. 1988; 66: 1054–1067. Publisher Full Text\n\nBérard A, Benninghoff C: Pollution-induced community tolerance (PICT) and seasonal variations in the sensitivity of phytoplankton to atrazine in nanocosms. Chemosphere. 2001; 45(4–5): 427–437. 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Bezdicek, and B.A. Stewart (eds.), Defining Soil Quality for a Sustainable Environment. SSSA Spec. Pub. No. 35. Soil Sci Soc Am Am Soc. Argon., Madison, WI. 1994; 3–21. Publisher Full Text\n\nElliott ET, Paustian K, Frey SD: Modeling the measurable or measuring the modelable: a hierarchical approach to isolating meaningful soil organic matter fractionations. In: Powlson, D.S., Smith, P., Smith, J.U. (Eds.), Evaluation of Soil Organic Matter Models. Springer-Verlag, NATO ASI Series. 1996; 161–179. Publisher Full Text\n\nFujita Y, Nakahara H: Effects of cultivation conditions on algal communities in paddy soils. Jap J Limnol. 1999; 60(1): 77–86.\n\nGaydon DS, Probert ME, Buresh RJ, et al.: Modelling the role of algae in rice crop nutrition and soil organic carbon maintenance. European Journal of Agronomy. 2012; 39: 35–43. Publisher Full Text\n\nGerson U: The associations of algae with arthropods. Rev Algol. 1974; 11: 18–41. Reference Source\n\nGoyal SK: Algae and the soil environment. Phykos. 1997; 36: 1–13.\n\nHargrove WW, Luxmore RJ: A New High-Resolution National Map of Vegetation Ecoregions Produced Empirically Using Multivariate Spatial Clustering. 1988. Reference Source\n\nHastings KL, Smith LE, Lindsay M, et al.: Microalgae counts raw data. F1000Research. 2014. Data Source\n\nHavlin JL, Beaton JD, Tisdale SL, et al.: Soil Fertility and Fertilizers. 6th Edition. Prentice Hall. Upper Saddle River, NJ. 1999; 499. Reference Source\n\nHeger TJ, Straub F, Mitchell EAD: Impact of farming practices on soil diatoms and testate amoebae: A pilot study in the DOK-trial at Therwil, Switzerland. European Journal of Soil Biology. 2012; 49: 31–36. Publisher Full Text\n\nHoffmann L: Algae of terrestrial habitats. Bot Rev. 1989; 55(2): 77–105. Publisher Full Text\n\nJohansen JR: Cryptogamic crusts of semiarid and arid lands of North America. J Phycol. 1993; 29(2): 140–147. Publisher Full Text\n\nKennedy AC, Papendick RI: Microbial characteristics of soil quality. 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Algal communities Vegetation. 1996; 125: 123–136. Publisher Full Text\n\nMalakar E, Kalita MC: A perspective towards development and commercialization of potential bga biofertilizers of Assam, North East India and carrier materials for BGA mass production and Inoculum development. Annals of Biological Research. 2012; 3(1): 814–828. Reference Source\n\nMartin E, Hine R: “Succession”. A Dictionary of Biology (6th ed.). Oxford University Press. 2008. Reference Source\n\nMcCann AE, Cullimore DR: Influence of pesticides on the soil algal flora. Residue Rev. 1979; 72: 1–31. Publisher Full Text\n\nMegharaj M, Singleton I, Kookana R, et al.: Persistence and effects of fenamiphos on native algal populations and enzymatic activities in soil. Soil Biol Biochem. 1999; 31(11): 1549–1553. Publisher Full Text\n\nMetting B: The systematics and ecology of soil algae. Bot Rev. 1981; 47(2): 195–312. Publisher Full Text\n\nMetting B, Rayburn W: Algal communities and soil microenvironments in an Eastern Washington silt loam. Soil Sci. 1979; 127(2): 74–78. Publisher Full Text\n\nMostafa FI, Helling CS: Impact of four pesticides on the growth and metabolic activities of two photosynthetic algae. J Environ Sci Health B. 2002; 37(5): 417–444. PubMed Abstract \| Publisher Full Text\n\nNeustupa J: Soil algae from marlstone-substratum based biotopes in the Nature park Džbán (Central Bohemia, Czech Republic) with special attention to the natural treeless localities. Algological Stud. 2001; 101: 109–120. Reference Source\n\nPan Y, Stevenson RJ: Gradient analysis of diatom assemblages in western Kentucky wetlands. Journal of Phycology. 1996; 32(2): 222–232. Publisher Full Text\n\nPandey JP, Tiwari A: Optimization of biomass production of Spirulina maxima. J Algal Biomass Utilization. 2010; 1: 20–32. Reference Source\n\nPankhurst C, Doube BM, Gupta V: Biological Indicators of Soil Health. CAB International, London, 1997. Reference Source\n\nPaoletti MG: Soil invertebrates in cultivated and uncultivated soils in North-East Italy. Redia. 1988; 71(2): 501–563. Reference Source\n\nPaoletti MG: Some unorthodox thoughts: what Western agriculture should learn from Chinese agriculture. Critical review in Plant Sciences. 1999; 18(3): 475–487. Publisher Full Text\n\nPaul EA, Clark FE: Soil Microbiology and Biochemistry. Academic Press, Inc. San Diego. 1989; 273. Reference Source\n\nPipe AE: Pesticide effects on soil algae and cyanobacteria. Bull Environ Contam Toxicol. 1992; 127: 95–170. Publisher Full Text\n\nPipe AE, Cullimore DR: An implanted slide technique for examining the effects of the herbicide Diuron on soil algae. Bull Environ Contam Toxicol. 1980; 24(1): 306–312. PubMed Abstract \| Publisher Full Text\n\nPringsheim EG: Pure cultures of Algae: their preparation & maintenance. CUP Archive. 1946. 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Publisher Full Text\n\nTsujimura S, Nakahara H, Ishida N: Estimation of soil algal biomass in salinized irrigation land: a comparison of culture dilution and chlorophyll a extraction methods. J Appl Phycol. 2000; 12(1): 1–8. Publisher Full Text\n\nvan Dam H, Mertens A, Sinkeldam J: A coded checklist and ecological indicator values of freshwater diatoms from the Netherlands. Netherlands Journal of Aquatic Ecology. 1994; 28(1): 117–133. Publisher Full Text\n\nWarkentin B: The changing concept of soil quality. J Soil Water Conserv. 1995; 50(3): 226–228. Reference Source\n\nWhitton BA: Soils and rice-field. in: Whitton B.A., Potts M. (Eds.). The Ecology of Cyanobacteria, their diversity in time and space. 2000; 233–255. Publisher Full Text\n\nYeates GW, Bongers T, de Goede RG, et al.: Feeding habits in soil nematodes families and genera-an outline for soil ecologists. J Nematol. 1993; 25(3): 315–331. PubMed Abstract \| Free Full Text\n\nZancan S, Trevisan R, Paoletti MG: Soil algae composition under different agro-ecosystems in North-Eastern Italy. Agriculture, Ecosystems and Environment. 2006; 112(1): 1–12. Publisher Full Text\n\nZenova GM, Shtina EA, Dedysh SN, et al.: Ecological relations of algae in biocenoses. Mikrobiologiya. 1995; 64(2): 121–133. Reference Source\n\nZurek L: The Influence of the Herbicides Lenacil and Pyrazon on the Soil Algae. Ekologia polska-Polish Pol J Ecol. 1981; 29(3): 327–342. Reference Source" }	[ { "id": "6723", "date": "24 Nov 2014", "name": "Matthew A. Bowker", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI reviewed Effect of microalgae application on soil algal species diversity, cation exchange capacity and organic matter after herbicide treatments by Hastings et al. I believe that I have an appropriate levels of expertise to determine whether or not it meets an acceptable scientific standard. This study adds a proprietary algal amendment to agricultural soils prior to cropping and measures changes in pH, Om, CEC, and the algal-cyanobacterial community. While the idea is interesting, and there is a long history of research on algal or cyanobacterial soil amendments, I cannot recommend this work due to various flaws, some rather severe. Major comments:Most problematic is that the experimental design is unreplicated. The algal product is added to 4 plots at different rates, with a 5th plot used as a control. Each plot has a high and low pH region. Measurements are conducted after application and again after 2 months. This would have been a much better designed experiment if there was at least some minimal replication (e.g. 3 plots of each type). The lack of replication really precludes most statistical analyses except perhaps for a before-after paired T-test analysis, and it is unclear to what degree apparent treatments differences arise simply because all of the plots are a little different in, e.g. pH. The formulation of hypotheses is weak regarding OM, CEC and pH. They can be inferred but are not stated. Also, the authors state that the primary purpose was to determine if the algal suspension improved species diversity of microalgae. Since this is obviously an applied study, readers will wonder why this is a goal worth attaining….isn’t the purpose of a soil conditioner the improvement of soil properties in a way that favors crop yield? Why would farmers care if there is more algal diversity in their soils? Figure 3 does not illustrate species diversity, it illustrates the proportion of isolates from the plots that fall into the broad taxonomic groupings of chlorophyte, cyanobacteria, and diatom. This may be related to evenness, but there is never any presentation of data on richness, so statements about diversity seem off target. Either the soil sampling technique is inadequately described or it is flawed. I understood that only 1 gram of soil was sampled from each plot, presumably a single sample due to its size. A superior sampling strategy would be to collect soil from multiple locations across the plot, pool and homogenize them, then conduct measurements on soil from the mixture. We cannot understand what is happening in an agricultural field based on 1 gram of soil from a single point in a heterogeneous field. Statistical analysis leaves much to be desired due to the experimental design. Before-after paired T-tests are used to detect changes in OM, pH or CEC. Presumably the authors included only the 4 plots amended with algae for this comparison. In doing this, the authors are ignoring their own design which amends algae at different rates, and it’s not clear how the control is used if at all. Also there are many reasons besides algal addition that properties of the field would change, notably the growth and harvest of a corn crop….how can they be distinguished? Presumably the algae were grown in a medium of some kind containing nutrients. Are they applied in a suspension of this medium? If so, how are effects of the algae separated from effects of the medium. A better design would also have a treatment applying the product autoclaved & filtered so that the effects of the medium alone without the algae. Table 2 is really not conducive to detecting any patterns in the data. The discussion claims that the algal amendment had positive effects on the soil environment. The data presented do not support this interpretation. Again, it is difficult to know if these patterns mean anything given the experimental design, but I see: in high pH soils there is an ambivalent effect on OM compared to controls, in low pH soils there is no difference in CEC among controls and amended plots, in high pH soils some algal-amended plots have higher CEC, and some have lower CEC compared to controls, and in high pH soils some algal amendment rates increase pH (in contrast to what the authors state in the abstract). I just don’t see evidence to support a clear argument that algal amendment improved soil properties for crops. Minor comments:Cyanobacteria are photosynthetic bacteria, not algae. If you plan to use the term algae to collectively include cyanobacteria, provide a definition. For example:….eukaryotic algae (chlorophytes and diatoms) and cyanobacteria (hereafter collectively referred to as “microalgae”). In the introduction, the authors state that the use of microalgae has recently been suggested in a paper from 2012. I don’t doubt that this is true, but the statement implies that this is a new idea. It is not. See Singh RM (1950). Define the V4 & V5 stages", "responses": [ { "c_id": "1205", "date": "06 Feb 2015", "name": "Maria McLeod", "role": "Author Response", "response": "We are grateful the reviewer has taken the time to read and comment. Our responses to the comments are found below. Major comments:“Most problematic is that the experimental design is unreplicated. The algal product is added to 4 plots at different rates, with a 5th plot used as a control. Each plot has a high and low pH region. Measurements are conducted after application and again after 2 months. This would have been a much better designed experiment if there was at least some minimal replication (e.g. 3 plots of each type). The lack of replication really precludes most statistical analyses except perhaps for a before-after paired T-test analysis, and it is unclear to what degree apparent treatments differences arise simply because all of the plots are a little different in, e.g. pH.”We were also concerned with the experimental replications as they were not feasible and for that reason it was decided that the study would focus on planting 4 rows per plot and taking 6 random samples from each plot to account for the non-homogeneity of the soil. Each plot was 0.5 squared acres. A total of 4 plots were used per treatment (one low pH, one high pH) plus one control. The total land used for this study was: 2.5 squared acres or 10,117.14 m2 equivalent to the area of 2 American football fields with a plant population of over 30,000. While replication of plots would have been ideal, it was not feasible or cost effective. Therefore 6 samples were taken from each plot for comparison purposes including those from a plot planted the previous year. This would be the equivalent of planting 6 smaller plots within the same area. In terms of comparison, the low pH and high pH regions were compared separately due to the non-homogeneity of the soil. The formulation of hypotheses is weak regarding OM, CEC and pH. They can be inferred but are not stated. Also, the authors state that the primary purpose was to determine if the algal suspension improved species diversity of microalgae. Since this is obviously an applied study, readers will wonder why this is a goal worth attaining….isn’t the purpose of a soil conditioner the improvement of soil properties in a way that favors crop yield? Why would farmers care if there is more algal diversity in their soils?The hypothesis was stated as two separate objectives. These can be read in the introduction: “The first objective of this study was to determine the effects of the application of a commercial proprietary suspension of microalgae (Chlorella sp. 1×10e3 cells per mL, Nannochloris sp. 1×10e3 cells per mL, Scenedesmus sp. 1×10e3 cells per mL) formulated to provide nutrients to indigenous soil microorganisms and facilitate microbial density and diversity, as well as to increase soil carbon.” “The secondary objective of this study was to determine the effects of GOgreen™ on %OM and CEC in experimental plots with different pH and to compare them with those obtained without GOgreen™ treatment.” As stated in the manuscript: “The results presented in this manuscript demonstrate that treatment with GOgreen™ has significant and measurable effects on soil OM content, CEC, pH and microalgae species diversity suggesting positive effects of this formulation on soil conditions, making them more suitable to sustain crops.” We are working on a second publication that will cover the effects of GOgreen™ on crop yield. It has been very recently demonstrated by other groups of investigators that microalgae are able to fix nitrogen in the soil and that cyanobacteria improve nitrogen (N), phosphorus (P), potassium (K), iron (Fe), and other mineral content in soil and facilitate better use of such minerals in plant growth promotion for enhanced crop production (Kumar et al., 2015). Figure 3 does not illustrate species diversity, it illustrates the proportion of isolates from the plots that fall into the broad taxonomic groupings of chlorophyte, cyanobacteria, and diatom. This may be related to evenness, but there is never any presentation of data on richness, so statements about diversity seem off target.Species diversity is by definition a measure of the diversity within an ecological community that incorporates both species richness (the number of species in a community) and the evenness of species' abundances or simply stated, “the number of different species that are represented in a given community (a dataset)”. The pie charts on figure 3 showed species diversity as total counts of species per group and it is clearly seen from the chart that the number of species within each category varied with treatment when compared to the untreated controls. Either the soil sampling technique is inadequately described or it is flawed. I understood that only 1 gram of soil was sampled from each plot, presumably a single sample due to its size. A superior sampling strategy would be to collect soil from multiple locations across the plot, pool and homogenize them, then conduct measurements on soil from the mixture. We cannot understand what is happening in an agricultural field based on 1 gram of soil from a single point in a heterogeneous field.As stated in the manuscript, the protocols followed were those implemented by the Irrigation Research Foundation (IRF). Their protocols indicate taking 6 different samples from different places in the plot for analysis. This is the protocol followed in this study. Statistical analysis leaves much to be desired due to the experimental design. Before-after paired T-tests are used to detect changes in OM, pH or CEC. Presumably the authors included only the 4 plots amended with algae for this comparison. In doing this, the authors are ignoring their own design which amends algae at different rates, and it’s not clear how the control is used if at all. Also there are many reasons besides algal addition that properties of the field would change, notably the growth and harvest of a corn crop….how can they be distinguished?All plots were included in the comparison. Due to the heterogeneity of the soil, a “before and after” comparison seems more representative of treatment especially because the controls were also heterogeneous when compared to the plots. A comparison between different doses only showed one characteristic worth annotation. This result is and was mentioned in paragraph 3 of page 9: “At this pH, treatment with GOgreen™ the previous year (plot 3) and with twice the recommended application rate (plot 4) resulted in a pH increase of 0.2 units two months after harvest when compared to the other plots.” As growth and harvest and the influence of GOgreen™ on corn are of interest to readers, these findings will be discussed in detail in the following manuscript. Readers who would like more information on harvest and yield resulting from these studies, (before the second publication is available online) are invited to contact the authors directly. Presumably the algae were grown in a medium of some kind containing nutrients. Are they applied in a suspension of this medium? If so, how are effects of the algae separated from effects of the medium. A better design would also have a treatment applying the product autoclaved & filtered so that the effects of the medium alone without the algae.Yes, as the third paragraph of the introduction stated: “The first objective of this study was to determine the effects of the application of a commercial proprietary suspension of microalgae (Chlorella sp. 1×10e3 cells per mL, Nannochloris sp. 1×10e3 cells per mL, Scenedesmus sp. 1×10e3 cells per mL)”. Since the product is sold as a commercial suspension, the product was applied in the same way a farmer would apply it after purchasing it. Autoclaving the product would have been detrimental to the live algae defeating the purpose of the study. Table 2 is really not conducive to detecting any patterns in the data.As the title indicates, table 2 describes “Treatment and applications for plots 1–4 and control during 2011”. There was no intention to provide patterns in the data for this table. The discussion claims that the algal amendment had positive effects on the soil environment. The data presented do not support this interpretation. Again, it is difficult to know if these patterns mean anything given the experimental design, but I see: in high pH soils there is an ambivalent effect on OM compared to controls, in low pH soils there is no difference in CEC among controls and amended plots, in high pH soils some algal-amended plots have higher CEC, and some have lower CEC compared to controls, and in high pH soils some algal amendment rates increase pH (in contrast to what the authors state in the abstract). I just don’t see evidence to support a clear argument that algal amendment improved soil properties for crops.CEC and OM are closely related to pH. Cation exchange is pH dependent by definition. Different crops require different pH values and different types of soil exhibit different CEC. As stated in the objectives, the purpose of this work was to show that it is possible to affect CEC and OM as well as pH when a live microalgae suspension is applied to the soil. The pH requirements for a particular type of soil would help a farmer make a decision regarding product use and timing. Minor comments:Cyanobacteria are photosynthetic bacteria, not algae. If you plan to use the term algae to collectively include cyanobacteria, provide a definition. For example: ...eukaryotic algae (chlorophytes and diatoms) and cyanobacteria (hereafter collectively referred to as “microalgae”).We agree with the reviewer. Prior to the use of molecular biology for identification, cyanobacteria were formerly called blue-green algae because they are photosynthetic and aquatic. We appreciate the comment and would like to clarify that we were unable to find the term Cyanobacteria associated with the term blue-green or algae in our write up of this manuscript except for text referenced from publications that are over 10 years old. In the introduction, the authors state that the use of microalgae has recently been suggested in a paper from 2012. I don’t doubt that this is true, but the statement implies that this is a new idea. It is not. See Singh RM (1950).We were unable to find Singh RM in references related to microalgae from 1950. We would appreciate it if the reviewer could provide the complete reference. Define the V4 & V5 stagesWe were unable to find V4 in the document. We were able to find V5 in a phrase on the first paragraph of page 4 as “V5 stage of plant growth as well as 4, 6, 7, and 8 weeks after emergence”. The term refers to a stage of plant growth. Leaf stages are usually described as “V” stages. V5 stage of plant growth is defined as the stage where the collar of leaf number 5 is visible. Once again, we would like to thank the reviewer for taking the time to read our manuscript and we respect the opinions expressed." } ] }, { "id": "9124", "date": "22 Jun 2015", "name": "Judith K. Brown", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title seems appropriate for the article content and the abstract provides a summary of the research aim, results, and major conclusion. The objective of the study was to determine the effect of GOgreen® on algae species diversity in a field heavily cultivated (farmed) and subjected to herbicide application, which is reported to diminish microalgal species diversity (‘assemblages’). Use of the product is purported to increase the content of green to blue-green algae, thereby increasing soil health with respect to pH and other parameters. The hypothesis is that the proprietary algal community preparations applied to the soil prior to cultivation of a corn crop would provide beneficial effects measureable in terms of pH, cation exchange capacity, organic matter content, and algal content (green, blue-green, diatoms). The second objective addressed the effect of the treatment on OM and CEC, which are pH dependent. The methodologies and analyses are well described and are appropriate for the research questions addressed pertaining to a proprietary microalgal product (GOgreen) applied through an irrigation system to corn plants (half acre plots) grown in soil previously treated with an herbicides and subjected to routine use for farming (consecutive planting/harvesting seasons). The premise is that the naturally occurring soil microalgal species, which contribute to soil health, are hindered by herbicide treatment. The algal species in treated soil sampled at a 6-inch depth were identified morphologically prior to the treatment, and at 4,6,7, and 8 weeks after seedling emergence, at V5 stage of plant growth, and at harvest. The experimental control consisted of adjacent plot of land that was not treated with the product. The conclusions appear to be justified based on the aim, methods, and results reported.The results suggested that treating soil with the product showed improvement in terms of organic matter content (increased when initial pH was greater than pH 8.0 but lower in soil of starting pH less than 8.0), cation exchange capacity, and increased diversity of certain (green and blue-green) microalgal species in soil samples monitored throughout the study. Some positive effects of the treatment were observed with respect to soil pH that is attributed to altered microalgal composition, and subsequently, with respect to CEC and OM. Initial pH readings in the plot were 8.3 and 6.8 in different parts of the field, respectively.\n\nThey conclude that treatment with GOgreen® resulted in improved OM content in the soil during the cropping season, but not longer than two months after harvest. Also, they conclude that increased OM was associated with differences in initial pH. The results are reasonably conclusive in favor of a transient modification in soil parameters due to the treatment with the proprietary microalgal preparations. The conclusions seem to indicate that the product must be applied at each planting. This is no different from the routine application of fertilizers and other conditioning agents used to improve soil structure and fertility, and so the product does appear to make a positive contribution to the soil for crop production (in this case, for maize). NOTE: the authors should double check the pH values – in Methods the low pH is reported at 6.8, but in the results they mention pH 6.9 instead. The data inform the aims/methods; the methods are appropriately detailed such that the experiments could be reproduced in another laboratory. No discrepancies or format issues are apparent. The manuscript appears suitable for indexation based on my understanding of the topic, and review of the methods, results, and conclusions.", "responses": [] } ]	1	https://f1000research.com/articles/3-281
https://f1000research.com/articles/3-279/v1	14 Nov 14	{ "type": "Opinion Article", "title": "Connecting undergraduate science education with the needs of today’s graduates", "authors": [ "Viviane Callier", "Richard H. Singiser", "Nathan L. Vanderford", "Viviane Callier", "Richard H. Singiser" ], "abstract": "Undergraduate science programs are not providing graduates with the knowledgebase and skills they need to be successful on today’s job market. Curricular changes relevant to today’s marketplace and more opportunities for internships and work experience during students’ secondary education would facilitate a smoother transition to the working world and help employers find graduates that possess both the hard and soft skills needed in the workplace. In this article, we discuss these issues and offer solutions that would generate more marketplace-ready undergraduates.", "keywords": [ "undergraduate education", "science curricula", "work experience", "job market", "job skills" ], "content": "Introduction\n\nThe premium for an undergraduate degree is high: compared to high school graduates, college graduates in Science, Technology, Engineering and Mathematics (STEM) fields earn on average $1.5 million more over their lifetime (Austin, 2014). This effect remains even after controlling for family background and other variables that could differentiate the population of students that pursue a college education from those who do not. Thus, attending college and studying a STEM field is still worth the cost (Daly & Bengali, 2014) despite the ever-increasing tuition rates, the increasing burden of student debt (Ernst, 2014), and the bad job market students encounter upon graduation (Weissman, 2014). Notwithstanding, successfully obtaining an education certainly does not guarantee success in today’s job market (Bersin, 2014).\n\nUndergraduate education is badly in need of reform. Receiving an education is not the same as receiving job training, and too many students graduate with heavy debt and are ill-equipped to thrive in today’s job market (Carpenter, 2014). The US Census Bureau has documented that many students cannot find jobs after graduation, and many of those who do find themselves employed in work that does not fully match their education/training. Students would be better served by an education that is integrated with the job market they will encounter post-graduation, and one that provides not only technical skills but also the soft skills that are most in demand by employers such as communication and interpersonal skills; decision-making skills; time and project management skills; problem-solving skills, and the ability to learn new skills quickly (The Association Of American Colleges and Universities, 2010; The Association Of American Colleges and Universities, 2013; Tugend, 2013; White, 2013). In other words, science training at the undergraduate level should move beyond rote memorization of facts and personal character building such as persistence, perseverance, or motivation; it needs to become specific and relevant to jobs.\n\nMost departments still use an old curriculum to teach traditional chemistry, biochemistry, biology, and molecular biology. Most students receive the same general curriculum no matter what they want as a career: find a job in industry, go to graduate school to do research, go to medical school to become a practicing physician, etc. As a consequence of undergraduate institutions doing a poor job of preparing students to be competitive for meaningful jobs upon graduation, many students pursue additional graduate training simply because they are not aware of other ways in which their undergraduate science degree could be used.\n\nCurrently, many agencies central to biochemistry and molecular biology have made curriculum recommendations. For example, the National Research Council has made some recommendations but these have not been widely implemented and miss the mark in terms of preparing highly functional, work-ready graduates, because they are too focused on traditional curricula and classroom-learning (2010). Although funding agencies, such as the National Science Foundation (NSF), push for education and outreach activities in the “broader impacts” criteria for grants, they have not sufficiently emphasized professional development of trainees specifically with respect to today’s job market. To reform undergraduate science education, we discuss below our suggestions of updating curricula and integrating work experience into programs.\n\n\nCurricular changes\n\nAt many universities, the current curricular model is outdated and employers frequently complain that graduates do not emerge with the skills they need (Dostis, 2013). Disciplines are largely compartmentalized for historical reasons, yet most creative and innovative work comes from bridging disciplines and using concepts and tools from a variety of fields to solve important problems.\n\nOne solution is to build in interdisciplinary topics within standard STEM courses in a way that will allow students the opportunity to explore current problems in environmental science, energy fields and/or public health. For example, green/sustainable chemistry—currently a central theme in all the divisions at the Environmental Protection Agency (EPA)—could be incorporated into traditional biochemistry curriculum. Green chemistry is an interdisciplinary topic and needs to be addressed from a variety of perspectives: chemical synthesis, environmental health, and the biochemistry and molecular biology of mechanisms of action. Evidence suggests that students show great interest in the research opportunities in green chemistry and risk assessment, and students themselves clearly are pushing for incorporating current issues in energy, environment and health into their core science curriculum (Goodman, 2009). These are excellent topics for teaching biochemistry and molecular biology students about how interdisciplinary life science topics interconnect with public health.\n\nCurrent research and marketplace issues are highly interdisciplinary, and thus, students should be trained in interdisciplinary work. Another example of this is in the collaboration between mathematicians and biologists to understand metabolic systems (e.g., folate metabolism, or insulin signaling) in cells. The function of the network is an emergent property that cannot be understood at the level of individual components. The response of metabolic networks to perturbations cannot be analyzed by verbal arguments; instead, it is necessary to describe the network using a system of differential equations. This allows researchers to study its dynamic behavior with simulations. The simulations will in turn suggest interesting predictions about network function to test experimentally in the lab. The feedback between experiment and theoretical modeling is a powerful approach to complex biological problems and is only possible when interdisciplinary teams work together.\n\nInterdisciplinary training in teams provides students the opportunity to develop soft skills such as communicating with researchers in different fields—each of which has specialized language and concepts. In addition, coursework in mathematical biology is an opportunity for STEM students to receive adequate training in quantitative skills (mathematics, statistics and data analysis) and computer programming. These skills are not only critical for pursuing a research career, but are also highly transferable skills that are valued by employers in a variety of fields.\n\nUndergraduate programs could also take lessons from innovative graduate school initiatives. A course co-organized by the Society for Cell Biology and the Keck Graduate Institute, and funded by the biotech company EMD Millipore, Inc., provides a “crash course” for 40 selected graduate students and postdoctoral fellows interested in transitioning to careers in the biotechnology industry. The course provides MBA-style training, professional development workshops, and a team-based project. Funding from EMD Millipore is a generous investment in the training of scientists that the company may ultimately recruit. The demand for such programs is extremely high and there is clearly a need for more programs like this because STEM graduate programs currently fail to prepare their students (or postdoctoral fellows) for jobs outside of academia. Similar programs could be established in the undergraduate setting to fill a similar gap. We are aware of some institutions that are moving in this direction. For example liberal arts colleges such as Mount Holyoke, which are traditionally not focused on job-training, are creating an entrepreneurial track and developing a program focused on environmental sustainability (Weir, 2014). Connecticut College, another liberal arts college, has created a program (Connecticut College’s Career Enhancing Life Skills) to help undergraduates identify and develop a career path starting from their first year in college and to establish connections with potential employers throughout their undergraduate career. In addition to helping students, in almost every context, enhancing the communication between potential employers and faculty could help identify the skills that are currently lacking in many of the graduates currently produced by universities and lead to productive dialogue about curricular changes to remedy this issue.\n\n\nWork experience\n\nIn addition to incorporating curricular changes, departments and institutions should be providing bridges to the workplace such as internships. These provide critical work experience leading to the development of skills that students cannot get in the classroom, such as firm-specific technical training, but also soft skills such as working collaboratively, facilitating group decision-making, serving customers, and sales/marketing. Internships and work experience also provide critical networking opportunities that may lead to job opportunities (job offers, referrals, recommendations, etc.). Some universities, such as the Ira A. Fulton Schools of Engineering at Arizona State University, have already created partnerships with industry for mentorship and internships. Urban universities could readily incorporate internships into their programs as they have the advantage of being surrounded with companies which can offer internship experiences; rural universities could create programs with willing companies that could help students with logistics such as transportation and housing. Ultimately, how better for undergraduates to obtain the real world working experience needed to successfully gain employment after graduation than by working in the real world as part of their education?\n\n\nConclusions\n\nIn today’s competitive job market, students need to emerge from their undergraduate STEM education with relevant technical skills as well as soft skills such as creativity, resourcefulness, intellectual curiosity, respect for others, ability to be self-directed yet able to work effectively as part of a team. Most importantly, they should emerge with a good understanding of the job options they have in a variety of sectors, work experience, and a network of professional contacts that will help them move forward in their careers with confidence, clarity and purpose.\n\nWe propose the following recommendations for changes to undergraduate STEM curriculum to better prepare students to thrive in the job market they will have to navigate upon graduation:\n\n1) Universities/departments need to update traditional core curricula to include interdisciplinary topics that highlight connections between the standard curriculum and current, real-world STEM issues. To achieve this, there are three levels of change that institutions could invoke; these levels increase in difficulty and impact both on the institution and on students, but ultimately these changes would add significant value to students’ career development.\n\nFirst, topics such as green chemistry and computational biology could be the focus of at least one lecture per semester in standard chemistry, biology/molecular biology, and/or biochemistry courses. This would be an easily change in the core curricula that would introduce students to topics and skills that directly apply to currently trending marketplace issues.\n\nSecond, STEM programs could encourage students to take non-science courses that are directly relevant to the job market. These courses could be taken as part of students’ elective coursework. We suggest that STEM programs should encourage students to take courses that would build business acumen (for example, courses on organizational behavior, leadership, entrepreneurship, strategy, and operations management); develop interdisciplinary teamwork skills through the integration of topics covering biochemistry/molecular biology, math, and computer programming/coding, public health; and lastly, enrich workplace readiness through career development topics including interviewing, resume building and networking. Universities could develop a “Preparing STEM Professionals” certificate program that would give students’ incentive to enroll in these types of courses.\n\nA third, stretch solution, would be for institutions to create entirely new courses that address the intersection of the standard core curricula with today’s most important global topics. Some institutions are taking steps in this direction. For example, the chemistry and biochemistry courses at California State University at Fullerton include such offerings as biotechnology: science, business, and society; environmental pollution and solutions; introduction to computational genomics; advanced computational biochemistry; and internships in chemistry and biochemistry. Other institutions should move in similar directions. As part of these courses, students should be given opportunities to work collaboratively on projects in interdisciplinary teams, as the ability to work as part of a team is highly valued by employers. Training in quantitative data analysis and programming—sorely lacking in too many undergraduate biology/chemistry/biochemistry programs—should also be emphasized.\n\nUltimately, building the interdisciplinary and “soft” skills employer’s desire should be the focus on these curricular changes. The curriculum should teach students to think critically and creatively about current and future problems that need solving and that will be valued by employers.\n\nThere are likely existing programs that are achieving the outcomes we are suggesting. It would be useful for publishers to coordinate a series of articles on this subject to build awareness of the curricular changes that are already being implemented in institutions across the country and to develop guidelines and best practices for universities as they reform and update their STEM curricula to make them work-ready.\n\n2) Universities should provide impactful opportunities and support for internships and work experience. It is through these types of experiences that students will truly gain the most useful work preparedness during their undergraduate career. Students will build real work skills and develop contacts that will be important for future employment. Perhaps the least challenging way to accomplish meaningful internships is for institutions to form formal partnerships with local or regional companies.\n\nMany internship programs have been developed within STEM programs. For example, the Virginia Commonwealth STEM Industry Internship Program links undergraduate STEM students to paid internship positions with companies throughout Virginia; the National Homeland Security-STEM Summer Internship Program provides undergraduate juniors and seniors the opportunity to work with homeland security professionals and researchers for up to ten weeks during the summer; and the University of Connecticut’s UConn-TIP Bioscience and STEM Summer Research Intern Program pairs students with University technology start-up companies for mentored summer research internships. These are shining examples of programs that could be emulated across all undergraduate institutions.\n\nTo further incentivize integrating work experience into undergraduate curricula, we believe that funding agencies, such as NSF and the National Institutes of Health, have a key role to play. In the same way that funding agencies have promoted education and outreach in the “broader impacts” criterion for grants, they should also emphasize the need for clear, actionable career development opportunities (in academic and non-academic settings) for students. For example, in addition to NSF funding Research Experiences for Undergraduates (REUs) which are largely at academic institutions, NSF and NIH could also organize bridging experiences for students to explore research in industry, the world of science policy, and careers in science writing and editing. Funding agencies could develop workforce innovation funding opportunities that could incentivize the creation of unique solutions to creating work experience for undergraduates and these novel programs could serve as models for other institutions. Ultimately, funding agencies could drive a culture of creating practical work experience as part of undergraduate education.\n\nAgain, some institutions have found unique ways to successfully incorporate work experience into undergraduate STEM curricula in a way that benefits both the institution and students. Publishers could commission articles from such programs across to demonstrate their success, highlight challenges faced in development of such initiatives, and to establish discussions that may lead to the development of guidelines and best practices for undergraduate internship programs.\n\nGiven the rising costs of a college education, it is imperative that students emerge with their degrees with skills relevant to the job market. Too many employers complain that they can’t find the right talent and too many graduates are un- or under-employed. Changes in the undergraduate education system—curricular changes and integrated work experience—could remedy this problem. We encourage institutions and organizations to discuss the success and challenges they have faced in implementing such changes to the undergraduate education experience.", "appendix": "Author contributions\n\n\n\nVC, RHS, and NLV conceived and prepared the manuscript and have approved the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nAustin J: What’s your science degree worth? Science. 2014. Reference Source\n\nBersin J: Growing Gap Between What Business Needs and What Education Provides. Forbes. 2014. Reference Source\n\nCarpenter B: Is Your Student Prepared for Life? The New York Times. 2014. Reference Source\n\nDaly MC, Bengali L: Is it still worth going to college? Federal Reserve Bank of San Francisco Economic Letters. 2014. Reference Source\n\nDostis M: Degree alone not enough to prepare grads for workforce. USA Today. 2013. Reference Source\n\nErnst J: Student loan debts top $1 trillion in US. Reuters. 2014. Reference Source\n\nGoodman S: “Green chemistry” movement sprouts in colleges, companies. The New York Times. 2009. Reference Source\n\nNational Research Council (US) Committee on Undergraduate Biology Education to Prepare Research Scientists for the 21st Century. Transforming Undergraduate Education for Future Research Biologists. 2010. Reference Source\n\nThe Association Of American Colleges And Universities. Raising the Bar: Employers’ Views on College Learning in the Wake of the Economic Downturn. 2010. Reference Source\n\nThe Association Of American Colleges And Universities. It Takes More than a Major: Employer Priorities for College Learning and Student Success. 2013. Reference Source\n\nTugend A: What it Takes to Make New College Graduates Employable. The New York Times. 2013. Reference Source\n\nWeir EH: “Innovation Hires” position MHC for the future. Mount Holyoke News and Events. 2014. Reference Source\n\nWeissman J: How bad is the job market for the college class of 2014? Slate. 2014. Reference Source\n\nWhite M: The Real Reason New College Grads Can’t Get Hired. Time. 2013. Reference Source" }	[ { "id": "6714", "date": "17 Nov 2014", "name": "Genevieve Newton", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting and timely article that presents viable solutions to some of the challenges being faced by higher education. The two main solutions presented - increasing the interdisciplinarity of STEM undergraduate curricula and providing more work experiences for students - are consistent with accepted high impact educational practices. The paper is quite brief, but nonetheless presents several concrete examples, and the authors rightfully encourage educators to share what they are doing in order to develop guidelines and best practices.In terms of the interdisciplinary curriculum argument, I would encourage the authors to broaden this even further to include disciplines such as sociology, political science, and psychology, as many of today's scientific issues (such as climate change and the challenge of feeding the planet) can be addressed from a multitude of different angles. In terms of the suggestion of increasing work experience for students, it would be helpful to see more discussion of the different approaches that can be taken in this regard. For example, consideration of co-operative programs, internships, externships, and course-embedded community engaged learning projects.Overall, this paper achieves the stated objective of describing strategies to connect undergraduate science education with the needs of today's graduates, and should prove informative to educators in STEM fields.", "responses": [ { "c_id": "1126", "date": "16 Dec 2014", "name": "Nathan Vanderford", "role": "Author Response", "response": "Dear Dr. Newton,We thank you for reviewing our article and for your comments. We agree that a number of interdisciplinary topics could (and should) be integrated into STEM curricula. We have limited the scope of our article to a detailed discussion of a few example topics and, via our suggestion within the article that others should report on their programs that have novel curricula, we hope to hear a variety of other examples that integrate a wide range of topics/disciplines into STEM curricula. This is also the case regarding your comment on additional methods for integrating work experience into STEM programs; we hope that our specific call for others to report on their programs leads to a number of other articles that share specifics about how institutions are integrating work experience into STEM curricula through a number of different methods including co-operative programs, inter/externships, etc. As such, we look forward to subsequent articles that can further address your comments.Thank you again for your time and comments.Viviane Callier, Richard H. Singiser, Nathan L. Vanderford" } ] }, { "id": "6825", "date": "03 Dec 2014", "name": "Laurence Lurio", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is more of an opinion piece than a research article. The idea of incorporating practical applications into STEM education is obviously a good idea, but the obstacles to implementation have not really been addressed, which is crucial. Statements such as \"science training at the undergraduate level should move beyond rote memorization of facts\" seem rather naive. No one, for a long time, has argued that undergraduate education should be rote memorization.", "responses": [ { "c_id": "1125", "date": "16 Dec 2014", "name": "Nathan Vanderford", "role": "Author Response", "response": "Dear Dr. Lurio,We thank you for reviewing our article and for your comments. As you note, the article does contain a few statements that could, arguably, be controversial, and as an opinion article, we feel that we are warranted in expressing our views on the current state of undergraduate education and on how we see ways to improve its future state. We agree with your point that “no one, for a long time, has argued that undergraduate education should be rote memorization” yet too often that is still what we see in the classroom, and it remains a problem. We also agree that there will be challenges/obstacles to implementing our recommendations and we believe that these may vary widely from institution to institution. We hope that by specifically mentioning in the article that publishers should help commission articles from programs that are implementing practical applications – such as updated curricula and the integration of work experience – that such articles would address associated challenges/obstacles, best practices, and success stories. As such, we believe that future articles will best address your point.Thank you again for your review. Viviane Callier, Richard H. Singiser, Nathan L. Vanderford" } ] }, { "id": "6827", "date": "18 Dec 2014", "name": "Marie-Claire Shanahan", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have hit on a very important issue here, and I am really pleased to see ongoing discussions of how undergraduate science education can be better tailored to students’ needs. The authors also introduce interesting and relevant examples of integrated curriculum topics (such as green chemistry) and of successful STEM career programs (such as Connecticut College’s Career Enhancing Life Skills). Given the importance of these issues, I would really encourage the authors to look at how the argument might be strengthened, in particular with support from empirical and peer-reviewed sources. The authors are clear in their views in a way that is appropriate for an opinion piece, but the factual claims that are made in service of the overall argument need better supporting sources. I understand that this is an opinion piece and am not suggesting an exhaustive review of the literature, just attention to a few key findings related to the arguments that are made here. For example, some claims are supported with weak evidence from opinion articles from media sources rather than empirical and peer-reviewed sources. E.g., “Too many students graduate with heavy debt and are ill-equipped to thrive in today’s job market” (p. 2) citing only Carpentier (2014), an opinion piece from the NYT that is supported with only an online survey from a job search website. Similarly the claim about the substance of employers' complaints about graduates (p. 2) is supported with a brief news article about a poll commissioned by an online homework help website. It would be important to at least examine the full report to ensure that the data is appropriate for making claims in an academic opinion piece. Other claims are made with no support at all, e.g., “Many students pursue additional graduate training simply because they are not aware of other ways in which their undergraduate science degree could be used.” (p. 2). A claim that quantitative skills are an example of “highly transferable skills that are valued by employers” is also unsupported. Off hand I don't know of any studies that specifically address quantitative skills as valued by employers but it could fall within the mismatch that Hernández‐March et al (2009) find in field-specific practical skills that employers desire but perceive that students lack. Sagen, Dallam and Laverty (2000) also find that quantitative training is related to job search success for undergraduates though they do not examine employers' desires directly. The paragraph that spans p. 2-3 describes several good examples such as Mount Holyoke and Keck Graduate Institute. It is a good illustration of beginning share examples and best practices, as the authors advocate in their conclusion. It could, again, be stronger if there were connections to some of the published case studies that try to assess claims like these in relation to specific programs, e.g., Junge et al (2010). Links to published case studies would also be very valuable in supporting the suggestion that publishers offer more venues for sharing best practices, challenges and successes. It would be important to acknowledge the venues that do exist, while also advocating for more. Overall, I commend the authors on tackling a very important issue and encourage their efforts to push this discussion forward. I think that their argument could be greatly strengthened, however, with better attention to at least a few key pieces of the literature in the area of workplace and employability programs in undergraduate education. They would find good support for their overall aims but also be able to make more nuanced arguments about how the important goal of improving undergraduate science education can be accomplished. To that end, here are a few pieces that might be of interest to the authors: Cranmer, S. (2006). Enhancing graduate employability: best intentions and mixed outcomes. Studies in Higher Education, 31(2), 169-184. A study of university departments examining their faculty members’ practices for teaching employability skills with attention to how well their goals are achieved. Coll, R. K., & Zegwaard, K. E. (2006). Perceptions of desirable graduate competencies for science and technology new graduates. Research in Science & Technological Education, 24(1), 29-58.Study of various stakeholders (including both employers and faculty) on what skills and competencies they prioritize, with a specific focus on “work-integrated learning”. Hernández‐March, J., Martín del Peso, M., & Leguey, S. (2009). Graduates’ skills and higher education: The employers’ perspective. Tertiary Education and Management, 15(1), 1-16.A large study of Spanish HR directors and company managers to examine what they see as required skills and what mismatches they perceive between undergraduate training and job requirements. Burke, V., Jones, I., & Doherty, M. (2005). Analysing student perceptions of transferable skills via undergraduate degree programmes. Active Learning in Higher Education, 6(2), 132-144.This is a case study of students at one undergraduate institution, examining their perceptions of the skills they have developed during their degree programs and how confident they are in their abilities to transfer those skills to a workplace environment. Sagen, H. B., Dallam, J. W., & Laverty, J. R. (2000). Effects of career preparation experiences on the initial employment success of college graduates. Research in Higher Education, 41(6), 753-767.A large study looking the factors that predict employment success of college graduates one month after graduation. Their regression model looked at a wide variety of factors from internship experiences to personal characteristics.Junge, B., Quiñones, C., Kakietek, J., Teodorescu, D., & Marsteller, P. (2010). Promoting undergraduate interest, preparedness, and professional pursuit in the sciences: an outcomes evaluation of the SURE program at Emory University. CBE-Life Sciences Education, 9(2), 119-132.A long term evaluation study of a summer research program that aimed to increase student preparedness for both graduate school and industry.", "responses": [ { "c_id": "1188", "date": "21 Jan 2015", "name": "Nathan Vanderford", "role": "Author Response", "response": "Dear Dr. Shanahan,We thank you for reviewing our article and for your comments. You clearly have a detailed understanding of the literature focusing on these issues. We have given your critique a great deal of thought, and we have decided to forgo submitting a revision to our article based on your comments primarily given the fact that F1000Research reviews can be independently cited. Ultimately, we feel that a revised version of the article would add no additional value beyond what is already captured in your critique. We therefore encourage readers to refer to and authors of subsequent work to reference your referee report. Thank you again for your review. Viviane Callier, Richard H. Singiser, Nathan L. Vanderford" } ] } ]	1	https://f1000research.com/articles/3-279
https://f1000research.com/articles/3-90/v1	11 Apr 14	{ "type": "Correspondence", "title": "Does the linear Sry transcript function as a ceRNA for miR-138? The sense of antisense", "authors": [ "Javier Tadeo Granados-Riveron", "Guillermo Aquino-Jarquin", "Javier Tadeo Granados-Riveron" ], "abstract": "Recently, the sex determining region Y (Sry) and the cerebellar degeneration-related protein 1 (CDR1as) RNA transcripts have been described to function as a new class of post-transcriptional regulatory RNAs that behave as circular endogenous RNA sponges for the micro RNAs (miRNAs) miR-138 and miR-7, respectively. A special feature of the Sry gene is its ability to generate linear and circular transcripts, both transcribed in the sense orientation. Here we remark that both sense (e.g. Sry RNA) and antisense (e.g. CDR1as) transcripts could circularize and behave as miRNAs sponges, and importantly, that also protein-coding segments of mRNAs could also assume this role. Thus, it is reasonable to think that the linear Sry sense transcript could additionally act as a miRNA sponge, or as an endogenous competing RNA for miR-138.", "keywords": [ "Sry RNA", "miR-138", "circRNA", "ceRNA", "sponge activity" ], "content": "\n\nCrosstalk involving RNA–RNA interactions adds a new dimension to our understanding of complex regulatory networks and offers profound implications for the elucidation of gene function1.\n\nMicroRNAs (miRNAs) are a type of endogenously expressed small regulatory non-protein-coding RNAs that negatively regulate gene expression by base-pairing (with imperfect complementarity) to miRNA response elements (MREs), which are usually located within the 3′-untranslated region (3′-UTR) of target RNA transcripts2. According to their number and location, it has become evident that a biological process may involve multiple miRNAs, and that a given gene may be regulated by more than one miRNA. Salmena et al. coined the term “competitive endogenous RNAs” (ceRNAs) to designate those transcripts that may cross-regulate each other by competing for shared miRNAs3. Multiple classes of non-coding RNAs (long ncRNAs) including circular RNA (circRNAs) and pseudogenes, and protein-coding mRNAs function as key ceRNAs and “super-sponges” to regulate the expression of mRNAs in plants and mammalian cells4.\n\nRecently, Hansen et al. and Memczak et al. described a new class of post-transcriptional regulatory RNAs that behave as circular endogenous RNA sponges (circRNAs) in two back-to-back papers published in Nature5,6. In both reports, the authors demonstrated that a ~1.5-kb single-stranded antisense circRNA molecule (human CDR1as or ciRS-7) containing multiple miR-7 binding sites densely arranged, acts as a natural miRNA sponge, by capturing complexes formed by miR-7/Ago2. Memczak et al. observed that human CDR1as expression in zebrafish impaired midbrain development, similar to knocking down miR-75.\n\nHansen et al. also showed that another circular RNA molecule, transcribed from the mouse Sry gene, could also act as an endogenous sponge. They noted that this transcript contains 16 binding sites for miR-138 and demonstrated in vitro that the Sry circRNA selectively “absorbs” this specific miRNA. Recently, Kartha and Subramanian asserted, based on the report by Hansen et al., that this Sry RNA is an antisense circular transcript that functions as a miRNAs sponge7. Although this apparently is a typographical error (antisense instead of sense), it was also referred as such in the original report by Memczak et al. in Nature. This suggests that the circular Sry transcript is, as occurs with the CDR1as sponge, an antisense circular RNA. Although it seems obvious that sponges are antisense to the miRNA they bind to, it should not be assumed that all circRNAs are transcripts in an antisense orientation to a protein coding gene, as occurs with CDR1as. A special feature of the Sry gene is that it can generate linear as well as circular transcripts depending on the use of alternative promoters (proximal vs distal)8. Capel et al. reported for the first time that the circular Sry RNA is derived from a sense sequence that consists of a single exon. This molecule is formed by the processing of a longer precursor transcript that contains one inverted repeat at each end. This unusual configuration promotes the formation of a stem-loop structure that facilitates the nucleophilic attack of a donor splicing site at the 3′ end to an acceptor site at the 5′ end, which results in its circularization8 (Figure 1). Thus, it can be asserted that this is, in fact, a circular sense Sry mRNA. Although the notion that the Sry circRNA is derived from an antisense transcript does not alter the interpretation of the results obtained by Hansen et al., we consider that this distinction is important, because it implies that both sense (e.g. Sry RNA) and antisense (e.g. CDR1as) transcripts could be circularized and act as RNA sponges, an observation which is not acknowledged by the authors of either of the original papers. Nevertheless, if the circular version of the Sry transcript can soak up miRNAs, can the Sry linear transcripts also do the same?\n\nAfter the Sry pre-RNA is transcribed, a stem-loop structure is created due to the presence of inverted repeats at the 5′ and 3′ ends. A normal splicing reaction takes place when the splice donor (SD) is attacked by a 2′-OH, presumably from a branch site adenosine residue (A) located in the intron, causing the first cleavage of the phosphodiester backbone. The newly formed 3′-OH at the SD, attacks the 5′-P at the splice acceptor (SA) site, resulting in excision of the intron and ligation of the circular exon of 1231 nucleotides. Modified from Capel et al.8.\n\nIn this respect, there is evidence that certain miRNAs may function by targeting sites in the 5′-UTR9 and open reading frame (ORF) regions of mRNAs10, suggesting that miRNAs may modulate gene expression by mechanisms different from canonical 3′-UTR target mRNA suppression. Binding of a miRNA to a ceRNA not only prevents that miRNA from binding to other MREs, but can also repress translation from the coding segment of the ceRNA11. A study of the pseudogene of the phosphatase and tensin homolog PTEN, PTENP1, provided the first experimental evidence for the cross-talk between coding and non-coding RNAs12. Tay et al. found that several endogenous protein-coding transcripts, such as serine incorporator 1 (SERINC1), vesicle-associated membrane protein associated protein A (VAPA), CCR4-NOT transcription complex and subunit 6-like (CNOT6L), act as PTEN ceRNAs, which regulate PTEN tumor suppressor levels in a miRNA-dependent manner12. This clearly suggests that mRNAs can function as ceRNAs and we propose that the mouse linear Sry sense transcript could also behave as a miRNA sponge, or as a ceRNA for miR-138. The extent to which other animal or human antisense or sense circRNAs also behave as miRNA sponges will doubtlessly be a subject of intense research. Shortly after the emergence of circRNAs, the first public circRNA database (circBase version 0.1) was developed by the Rajewsky laboratory as a compendium of thousands of circRNAs sequences that are expressed in eukaryotic cells. Access to this resource allows us to use the information in order to validate those circRNAs that are probably involved in many important cellular processes. Nevertheless, the precise molecular mechanisms that underlie post-transcriptional repression by circRNAs remain still largely unknown, but their discovery demonstrates the importance of this distinct type of non-protein-coding regulatory RNAs for the elucidation of gene function. Moreover, due to their longer half lives in vivo, circRNAs may possess a great potential for therapeutic intervention. Thus, manipulating miRNA function, either by mimicking or inhibiting ceRNAs implicated in several disorders such as cancer, could provide a novel strategy to interfere with disease initiation and/or progression. The antisense modulation of circRNAs/ceRNA→miRNAs→mRNAs→protein regulatory networks could offer ingenious decoy combinations (antisense technology) as well as delivery platforms for concurrently target multiple miRNAs in abnormal or undesired conditions13.", "appendix": "Author contributions\n\n\n\nJTGR and GAJ contributed extensively to this work and were involved in the critical revision of the manuscript. Both authors have agreed to the final version of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nWork in the group’s lab is supported by grants CB-168661 from the Mexican Council of Sciences and Technology (CONACyT) and Mexican Federal Funds (HIM/2012/010-SSA 1017).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nVidal M, Cusick ME, Barabasi AL: Interactome networks and human disease. Cell. 2011; 144(6): 986–98. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBartel DP: MicroRNAs: target recognition and regulatory functions. Cell. 2009; 136(2): 215–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSalmena L, Poliseno L, Tay Y, et al.: A ceRNA hypothesis: the Rosetta Stone of a hidden RNA language? Cell. 2011; 146(3): 353–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi JH, Liu S, Zhou H, et al.: starBase v2.0: decoding miRNA-ceRNA, miRNA-ncRNA and protein-RNA interaction networks from large-scale CLIP-Seq data. Nucleic Acids Res. 2014; 42(1): D92–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMemczak S, Jens M, Elefsinioti A, et al.: Circular RNAs are a large class of animal RNAs with regulatory potency. Nature. 2013; 495(7441): 333–8. PubMed Abstract \| Publisher Full Text\n\nHansen TB, Jensen TI, Clausen BH, et al.: Natural RNA circles function as efficient microRNA sponges. Nature. 2013; 495(7441): 384–8. PubMed Abstract \| Publisher Full Text\n\nKartha RV, Subramanian S: Competing endogenous RNAs (ceRNAs): new entrants to the intricacies of gene regulation. Front Genet. 2014; 5: 8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCapel B, Swain A, Nicolis S, et al.: Circular transcripts of the testis-determining gene Sry in adult mouse testis. Cell. 1993; 73(5): 1019–30. PubMed Abstract \| Publisher Full Text\n\nOrom UA, Nielsen FC, Lund AH: MicroRNA-10a binds the 5'UTR of ribosomal protein mRNAs and enhances their translation. Mol Cell. 2008; 30(4): 460–71. PubMed Abstract \| Publisher Full Text\n\nTay Y, Zhang J, Thomson AM, et al.: MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation. Nature. 2008; 455(7216): 1124–8. PubMed Abstract \| Publisher Full Text\n\nPoliseno L, Salmena L, Zhang J, et al.: A coding-independent function of gene and pseudogene mRNAs regulates tumour biology. Nature. 2010; 465(7301): 1033–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTay Y, Kats L, Salmena L, et al.: Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs. Cell. 2011; 147(2): 344–57. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang Z: The concept of multiple-target anti-miRNA antisense oligonucleotide technology. Methods Mol Biol. 2011; 676: 51–7. PubMed Abstract \| Publisher Full Text" }	[ { "id": "5178", "date": "19 Jun 2014", "name": "Gordon Carmichael", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article presents a short summary and review of recent work on circular RNAs and on competing endogenous RNAs, which can sequester and “sponge” microRNAs. A point made by the authors is that both sense and antisense RNAs from the same genomic region can act as miRNA sponges and that linear transcripts from the same regions that produce circular RNAs can themselves sequester miRNAs. Overall, I found the article to be provocative, though not novel. During the past several years there have appeared a number of papers describing circular RNAs of various sorts (for example, stable introns and circles resulting from splicing events commonly promoted by strong secondary structures flanking exons). There has also been a wealth of published work suggesting or showing that abundant RNAs can bind miRNAs and thus reduce their availability for the regulation of some mRNAs. Thus, this paper breaks little new ground, though it does point out that many genomic regions express both sense and antisense transcripts, and each of these can affect miRNA availability. To me, the area covered by this paper is a very interesting and important one, and it is of value to present and discuss new concepts in gene regulation. Thus, I think this manuscript would be improved by:More clearly summarizing the ceRNA field. Discussing a bit more the well known fact that sense and antisense transcripts are commonly expressed in cells, though not necessarily from the same locus at the same time. Mentioning the several ways that stable circular RNAs can be produced. Finally, discussing the issue lacking from the current version that, in order to effectively act as a miRNA sponge, an RNA must not only be stable, but also in the proper cellular compartment and containing a molar concentration of miRNA binding sites that is high enough to sequester a biologically significant fraction of the endogenous miRNA of interest.", "responses": [ { "c_id": "1068", "date": "06 Nov 2014", "name": "Guillermo Aquino-Jarquin", "role": "Author Response", "response": "1. More clearly summarizing the ceRNA field. We are grateful to Dr. Gordon Carmichael for all the comments made to our manuscript. A recently discovered molecular mechanism, named Competing Endogenous RNA (ceRNA) effect, has highlighted the importance of indirect interactions among transcript RNAs competing for the same pool of miRNAs 1. ceRNAs share one or more miRNA response elements (MREs) and compete for a restricted pool of common miRNAs. Thus, ceRNAs form complex regulatory networks of miRNAs and MRE-containing transcripts, both protein-coding and noncoding, which ensure a tight control of many biological processes. Aberrant expression of central nodes of such ceRNA networks may cause a disturbance that could contribute to disease pathogenesis 2. A discussion along these lines has been included in our new submission. 2. Discussing a bit more the well known fact that sense and antisense transcripts are commonly expressed in cells, though not necessarily from the same locus at the same time. CDR1as is a circRNA that functions as a sponge for miR-7, deriving from an antisense transcript of the CDR1 protein-coding gene. On the other hand, the transcript of the male sex-determining gene Sry, transcribed in sense orientation 3, is a second circRNA proposed to act as a sponge, in this case, for miR-138. However, Memczak et al. stated that “Perhaps the best known circRNA is antisense to the mRNA transcribed from the SRY (sex-determining region Y) locus and is highly expressed in testes” 4. Recently, Kartha and Subramanian asserted, based on the report by Hansen et al., that this Sry RNA is an antisense circular transcript that functions as a miRNAs sponge 5. Although this apparently is a typographical error (antisense instead of sense), it was also referred as such in the original report by Memczak et al. in Nature4. This would suggest that the circular Sry transcript is, as occurs with the CDR1as sponge, an antisense circular RNA. Although it seems obvious that sponges are antisense to the miRNA they bind to, it should not be assumed that all circRNAs are transcripts in an antisense orientation to a protein coding gene, as occurs with CDR1as. We conducted an in silico analysis taking the antisense sequence of the Sry mRNA to make a search for potential binding sites for miR-138, using the fuzzy bioinformatics tool (EMBOSS Suite) and we found none. When we used the Sry ORF (GenBank NM_011564) for the miR-138 binding site (CACCAGCA), we found 14 potential sites which is consistent with the number of sites found by Hansen and colleagues. For this reason, we remark in our current submission that both sense and antisense Sry transcripts could circularize and behave as miRNAs sponges, and importantly, that protein-coding segments of Sry mRNA could also assume this role. To our knowledge, there are no reports of a circRNA whose sequence in sense and antisense orientation possesses the ability to function as miRNA sponge. On the other hand, a particularly interesting family of non-protein coding RNAs consists of natural antisense transcripts (NATs). NATs are ncRNAs transcribed from the opposite strand of a coding gene and are capable of regulating the expression of their sense gene pair or of several related genes 6. Genomic loci that express NATs are highly abundant and sense/antisense (SAS) transcript pairs tend to be co-expressed. The most comprehensive studies predict that in human and mice 40-72% of all transcriptional units show evidence of bi-directional transcription 7. The regulatory activity of SAS pairs in human tissues has been postulated on protein expression at different levels, such as alternative splicing, post-transcriptional regulation, transport and epigenetic imprinting as well as transcriptional and translational interference through annealing to complementary sequences 8. As requested, we have included this information in the version of our submission. 3. Mentioning the several ways that stable circular RNAs can be produced. Recent bioinformatic and experimental analyses have identified thousands of circRNAs in the mammalian transcriptome, suggesting that circRNAs may in fact represent a new class of ceRNA regulators 9. These circRNAs are produced mainly through a type of alternative RNA splicing named ‘back-splicing’, in which a splice donor splices to an upstream acceptor rather than a downstream acceptor 10. Recently Guo et al. suggested that this would be the way in which generates most, if not all, cellular circRNAs. Please find a relevant discussion in the new version. 4. Finally, discussing the issue lacking from the current version that, in order to effectively act as a miRNA sponge, an RNA must not only be stable, but also in the proper cellular compartment and containing a molar concentration of miRNA binding sites that is high enough to sequester a biologically significant fraction of the endogenous miRNA of interest. Increasing experimental evidence supports the hypothesis that multiple non-coding RNA species, including small non-coding RNAs, pseudogenes, lncRNAs and circular RNAs (circRNAs) may possess ceRNA activity 9. The effectiveness of a ceRNA would depend on the number of miRNAs that it can ‘‘absorb’’ This, in turn, would depend on the ceRNA’s accessibility to miRNA molecules, which is influenced by its subcellular localization and its interaction with RNA-binding proteins. Furthermore, the specific cellular context in which the ceRNA is expressed would also impact its overall influence because not all microRNAs are present ubiquitously and at all times 11. We have included a discussion of the subject in the current version. References 1. Bosia C, Pagnani A, Zecchina R: Modelling Competing Endogenous RNA Networks. PLoS One. 2013; 8 (6). PubMed Abstract \| Free Full Text \| Publisher Full Text \| Reference Source 2. Karreth FA, Ala U, Provero P, Pandolfi PP: Pseudogenes as competitive endogenous RNAs: target prediction and validation. Methods Mol Biol. 2014; 1167: 199-212 PubMed Abstract \| Publisher Full Text \| Reference Source 3. Capel B, Swain A, Nicolis S, Hacker A, et al.: Circular transcripts of the testis-determining gene Sry in adult mouse testis. Cell. 1993; 73 (5): 1019-1030 PubMed Abstract \| Publisher Full Text \| Reference Source 4. Memczak S, Jens M, Elefsinioti A, Torti F, et al.: Circular RNAs are a large class of animal RNAs with regulatory potency. Nature. 2013; 495 (7441): 333-338 PubMed Abstract \| Publisher Full Text \| Reference Source 5. Kartha RV, Subramanian S: Competing endogenous RNAs (ceRNAs): new entrants to the intricacies of gene regulation. Front Genet. 2014; 5 (8). PubMed Abstract \| Free Full Text \| Publisher Full Text \| Reference Source 6. Khorkova O, Myers AJ, Hsiao J, Wahlestedt C: Natural antisense transcripts. Hum Mol Genet. 2014; 23 (R1): R54-R63 PubMed Abstract \| Free Full Text \| Publisher Full Text \| Reference Source 7. Werner A, Cockell S, Falconer J, Carlile M, et al.: Contribution of natural antisense transcription to an endogenous siRNA signature in human cells. BMC Genomics. 2014; 15 (19). PubMed Abstract \| Free Full Text \| Publisher Full Text \| Reference Source 8. Lapidot M, Pilpel Y: Genome-wide natural antisense transcription: coupling its regulation to its different regulatory mechanisms. EMBO Rep. 2006; 7 (12): 1216-1222 PubMed Abstract \| Free Full Text \| Publisher Full Text \| Reference Source 9. Tay Y, Rinn J, Pandolfi PP: The multilayered complexity of ceRNA crosstalk and competition. Nature. 2014; 505 (7483): 344-352 PubMed Abstract \| Free Full Text \| Publisher Full Text \| Reference Source 10. Guo JU, Agarwal V, Guo H, Vartel DP: Expanded identification and characterization of mammalian circular RNAs. Genome Biol. 2014; 15 (7): 409 PubMed Abstract \| Free Full Text \| Publisher Full Text \| Reference Source 11. Salmena L, Poliseno L, Tay Y, Kats L, et al.: A ceRNA hypothesis: the Rosetta Stone of a hidden RNA language?. Cell. 2011; 146 (3): 353-358 PubMed Abstract \| Free Full Text \| Publisher Full Text" } ] }, { "id": "4478", "date": "24 Oct 2014", "name": "Amy Pasquinelli", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis short commentary points out that both sense and antisense transcripts with miRNA target sites could serve to titrate the miRNAs and, thus, release other target RNAs from repression. Recently, there has been much interest in these so called competing endogenous RNAs (ceRNAs) as a form of regulating miRNA activity. While ceRNA activity has been attributed to pseudogenes and transcripts that circularize (circRNAs), theoretically any RNA, including protein coding mRNAs, could serve this function. The correspondence proposes that the linear Sry sense, as well as the previously reported antisense, transcript could function as sponges for miR-138. A major criticism of the proposal that ceRNA activity plays a function in regulating miRNA availability for target regulation is the consideration of cellular RNA concentrations. In many cases, the ceRNAs, even the circRNAs which can be more stable, are expressed at levels far below the miRNA or even its target mRNA. In fact, after this correspondence by Granados-Riveron & Aquino-Jarquin was originally published in April 2014, the hypothesis that ceRNAs can alter miRNA function in vivo was rigorously tested and the conclusion was published in May that most ceRNAs simply are not abundant enough to act as competitive inhibitors of miRNA binding to target mRNAs (Denzler et al., 2014). Thus, the authors need to include discussion of this concern over how generally miRNA activity might actually be affected by the presence of any kind of ceRNA, sense, antisense or circular.", "responses": [ { "c_id": "1069", "date": "06 Nov 2014", "name": "Guillermo Aquino-Jarquin", "role": "Author Response", "response": "This short commentary points out that both sense and antisense transcripts with miRNA target sites could serve to titrate the miRNAs and, thus, release other target RNAs from repression. Recently, there has been much interest in these so called competing endogenous RNAs (ceRNAs) as a form of regulating miRNA activity. While ceRNA activity has been attributed to pseudogenes and transcripts that circularize (circRNAs), theoretically any RNA, including protein coding mRNAs, could serve this function. The correspondence proposes that the linear Sry sense, as well as the previously reported antisense, transcript could function as sponges for miR-138. A major criticism of the proposal that ceRNA activity plays a function in regulating miRNA availability for target regulation is the consideration of cellular RNA concentrations. In many cases, the ceRNAs, even the circRNAs which can be more stable, are expressed at levels far below the miRNA or even its target mRNA. In fact, after this correspondence by Granados-Riveron & Aquino-Jarquin was originally published in April 2014, the hypothesis that ceRNAs can alter miRNA function in vivo was rigorously tested and the conclusion was published in May that most ceRNAs simply are not abundant enough to act as competitive inhibitors of miRNA binding to target mRNAs (Denzler et al., 2014). Thus, the authors need to include discussion of this concern over how generally miRNA activity might actually be affected by the presence of any kind of ceRNA, sense, antisense or circular. Firstly, we thank Dr Pasquinelli for her insightful comments. Secondly, we would like to clarify that the previously reported circular mouse Sry transcript reported by Capel et al. 1, now known to behave a sponge for mir-138 2,3, is, in fact, not derived from an antisense transcript but from a sense transcript, whose expression is directed by a promoter which is different to the promoter of the canonical linear Sry transcript involved in mammalian sex- determination. To the best of our knowledge, no antisense transcript of the murine Sry gene has been reported and a recent search of our own in publicly available EST data supports this assertion. Actually, one of our motivations for publish this piece of correspondence was to clarify this misconception (that the circular Sry RNA is an antisense transcript), which was first asserted in the report by Memczak et al. in Nature3 and later reproduced in a review of the subject by Kartha and Subramanian.4We agree that recent findings by Denzler et al. question the biological relevance of ceRNAs in terms of the abundance of these molecules which would be required to induce derepression of the targets of specific miRNAs 5. However, Memczak et al. and Hansen et al. shown that miRNA sponges selectively bind miRNAs forming complexes with Ago proteins, which raises the possibility that ceRNAs modulate gene expression not only by capturing miRNAs but also through the depletion of the pool of available effector molecules of the miRNA pathway. Additionally, Denzel et al. based their calculations for target abundance on sites present in transcriptome 3´UTRs, however, they were unable to rule out that unidentified highly abundant and regulated non coding RNAs (including circRNAs) might substantially contribute to the pool of available binding sites, a limitation acknowledged in their paper 5. This may be of particular importance in the adult testis, which express the circular Sry transcript and also has been shown to provide a permissive environment for transcription initiation, a phenomenon that has been called \"transcriptional promiscuity\" 6. Denzel et al. also state that their findings in liver can be generalized to other tissues and disease states, given that target abundance did not show large changes in the presence of insulin signaling or liver disease, conditions know to modify gene expression in hepatocytes. However, the authors also discuss that during cellular processes such as differentiation (like the spermatogenesis in the adult testis), expression of coding and non-coding RNAs changes dramatically, potentially making these systems more amenable to ceRNA-mediated gene regulation 5. We agree with Dr Pasquinelli in respect to the need of a discussion on the proposed mechanisms for ceRNAs action and their caveats in light of the findings by Denzel et al. and therefore, the new version of our correspondence includes such discussion. References 1. Capel B, Swain A, Nicolis S, Hacker A, et al.: Circular transcripts of the testis-determining gene Sry in adult mouse testis. Cell. 1993; 73 (5): 1019-1030 PubMed Abstract \| Publisher Full Text \| Reference Source 2. Hansen TB, Jensen TI, Clausen BH, Bramsen JB, et al.: Natural RNA circles function as efficient microRNA sponges. Nature. 2013; 495 (7441): 384-388 PubMed Abstract \| Publisher Full Text \| Reference Source 3. Memczak S, Jens M, Elefsinioti A, Torti F, et al.: Circular RNAs are a large class of animal RNAs with regulatory potency. Nature. 2013; 495 (7441): 333-338 PubMed Abstract \| Publisher Full Text \| Reference Source 4. Kartha RV, Subramanian S: Competing endogenous RNAs (ceRNAs): new entrants to the intricacies of gene regulation. Front Genet. 2014; 5: 8 PubMed Abstract \| Free Full Text \| Publisher Full Text \| Reference Source 5. Denzler R, Agarwal V, Stefano J, Bartel DP, et al.: Assessing the ceRNA hypothesis with quantitative measurements of miRNA and target abundance. Mol Cell. 2014; 54 (5): 766-776 PubMed Abstract \| Publisher Full Text \| Reference Source 6. Schmidt EE: Transcriptional promiscuity in testes. Curr Biol. 1996; 6 (7): 768-769 PubMed Abstract \| Publisher Full Text \| Reference Source" } ] } ]	1	https://f1000research.com/articles/3-90
https://f1000research.com/articles/3-268/v1	06 Nov 14	{ "type": "Case Report", "title": "Case Report: Rare occurrence of Pseudomonas aeruginosa osteomyelitis of the right clavicle in a patient with IgA nephropathy", "authors": [ "Aishwarya Damodaran", "Anusha Rohit", "Georgi Abraham", "Sanjeev Nair", "Anand Yuvaraj", "Aishwarya Damodaran", "Georgi Abraham", "Sanjeev Nair", "Anand Yuvaraj" ], "abstract": "We describe the case of a 47 year old patient with proven primary IgA nephropathy who presented with osteomyelitis of the medial end of the right clavicle. The patient was not on immunosuppressive medications. He underwent aspiration curettage and CT scan of the clavicle which yielded pus that grew Pseudomonas aeruginosa. Following treatment with appropriate antibiotic therapy the patient presented a complete recovery of the lesion with no loss of renal function. This case highlights the importance of positive cultures in the choice of the appropriate therapy in an extremely rare case of an immunocompetent patient with osteomyelitis of the clavicle.", "keywords": [ "Pseudomonas aeruginosa", "osteomyelitis", "IgA nephropathy", "antibiotics" ], "content": "Introduction\n\nOsteomyelitis of the clavicle is an extremely rare occurrence with an incidence of less than 1% in mixed age population, with Staphylococcus aureus being the most commonly isolated organism1,2. Patients often have a history of immunosuppression or invasive procedures such as tracheostomy or subclavian vein catheterisation. Here we report the case of a 47 year old man with IgA nephropathy who developed osteomyelitis of the medial end of right clavicle caused by Pseudomonas aeruginosa.\n\n\nCase description\n\nA 47 year old South Asian male teacher presented to our institute in June 2012 with a diagnosis made elsewhere of accelerated hypertension and acute left ventricular failure following a recent anterior wall myocardial infarction. He was admitted to hospital for further cardiac management, and a nephrology consultation was sought for renal insufficiency (serum creatinine 1.9 mg/dl). The presence of an active urinary sediment (urine albumin 3+ by dipstick and microscopic hematuria) necessitated a renal biopsy which showed IgA nephropathy (Figure 1) with an Oxford Pathological score of M1E0S1T1, 5/10 glomerular sclerosis and 30% IF/TA with hypertensive changes in blood vessels (Figure 2). The patient was initiated on olmesartan 20 mg/day and prednisolone 50 mg/day and a follow up for management of proteinuria was advised.\n\nHe presented again 7 months later with swelling pain and erythema of the right clavicle that had been present for 2 months. He was evaluated for these complaints at another centre before presentation to ours and was diagnosed with synovitis of the medial end of the right clavicle. Ultrasound guided aspiration of fluid from the swelling and analysis of bacterial cultures in an external laboratory did not reveal any organism. The patient was subsequently treated with non steroidal anti inflammatory drugs and prednisolone for 1 week.\n\nPhysical examination following admission at our centre revealed a normal body mass index (BMI) and vitals with an otherwise unremarkable systemic examination. Local examination of the right clavicle revealed a swelling of 2×3 cm with erythema, induration, warmth and tenderness with a firm consistency. The skin did not show discharging sinuses. Laboratory investigations revealed the following: white blood cells (WBC) count 12,400 cells/mm3 with predominant polymorphonuclear cells, platelet count 290,000/mm3, erythrocyte sedimentation rate (ESR) 93 mm/hr, urea 42 mg/dl and serum creatinine 1.5 mg/dl. Liver function tests revealed: serum glutamic oxaloacetic transaminase (SGOT) 69 IU, serum glutamic-pyruvic transaminase (SGPT) 44 IU, alanine aminotransferase (ALP) 130 kU, total protein 7.3 g/dl serum albumin 3.3 g/dl. Serology for HIV, hepatitis B and the venereal disease laboratory test (VDRL) were negative, anti-streptococcal antibody (ASO) was titre-negative, and C3 and C4 levels were within the normal range.\n\nUrine analysis showed 1+ albumin by dipstick, and microscopic examination of the urine showed 6–8 WBC/high power field (hpf), 2–3 epithelial cells/hpf, 4–6 RBCs/hpf and occasional granular casts. Sonography of the abdomen showed bilateral normal sized kidneys with increased echogenicity and maintained corticomedullary differentiation. Ultrasound guided aspirate of the pus from the right clavicle was cultured using BacT Alert 120 (bioMerieux, France) which showed Gram-negative and oxidase-positive rods. This suggested the presence of P. aeruginosa that was found to be sensitive to cefoperazone and sulbactam, using Vitek Compact II (bioMerieux, France). Repeated staining for Acid Fast Bacilli was negative. Tuberculosis (TB) PCR analysis and staining for fungal elements were negative. CT scan of the clavicle showed cortical and subcortical irregularity with soft tissue swelling of the medial end of right clavicle suggestive of osteomyelitis. Curettage and lavage of the site was followed by a gentamicin impregnated dressing (Septocoll E, Biomet Deutschland GmbH, Berlin), and histopathological examination of the sequestrum revealed non-caseating granulomas surrounded by many neutrophils, lymphocytes and plasma cells, suggestive of chronic osteomyelitis. The patient was begun on intravenous cefoperazone and sulbactum 1.5 g BD as per the sensitivity report for a period over 3 weeks.\n\n\nDiscussion\n\nOsteomyelitis most commonly involves the metaphyses of long bones and its occurrence in the clavicle as a primary infection is extremely rare1. The incidence of clavicular osteomyelitis in mixed adult population is less than 1% with 7% incidence in paediatric population3, and in adults is almost always due to prior trauma or invasive procedures in close proximity to the sternoclavicular area4. Examples of medical procedures that can cause osteomyelitis include tracheostomy, sternotomy or subclavian vein catheterisation, and osteomyelitis is often associated with immunosuppression therapy. Even in such cases the most common organism causing infection in more than 95% of the cases is S. aureus. In this case P. aeruginosa was the organism identified5.\n\nP. aeruginosa is an aerobic, motile, non-fermenting Gram-negative bacillus which produces a biofilm. It is usually regarded as a trivial commensal of the skin, mucosa and intestinal tract, but on occasions it can be the cause of severe hospital-acquired infections. Osteoarticular infections generally include pelvic and vertebral infections in patients with primary or secondary immunodeficiency, prior to prolonged broad spectrum antibiotic therapy, vascular insufficiency, intravenous drug abuse or other invasive procedures6,7 and, when secondary to Pseudomonas spp, are associated with a greater risk of recurrence and amputation6. Our patient had an Ig A nephropathy treated with low dose steroid therapy for only a short while (1 week). He presented evidence of chronic renal changes such as tubular atrophy, interstitial fibrosis and blood vessel changes as shown by the biopsy. As there was no other secondary cause detected we presumed that this was a primary IgA nephropathy. The natural course of IgA nephropathy ranges from benign non-progressive disease to end stage renal failure which occurs in 15–40% of the patients over a span of 10–20 years7.\n\nThe presence of osteomyelitis did not lead to rapid deterioration of renal function, as appropriate diagnosis and antimicrobial treatments were initiated without significant delay. As our patient had insignificant proteinuria, it is possible that his renal function deterioration would be slow as compared to patients with IgA nephropathy and heavy proteinuria.\n\nThere have been reports of reversals of secondary IgA nephropathies which developed post-chronic osteomyelitis after treating the infection8. But in this case, the diagnosis of IgA nephropathy was made 7 months prior to the onset of osteomyelitis. In this patient, there was no evidence to suggest a correlation between IgA nephropathy and profound renal insufficiency causing immunosuppression which led to osteomyelitis.\n\nIn conclusion, we report a rare occurrence of osteomyelitis of the clavicle due to P. aeruginosa in a non-immunosuppressed patient with chronic kidney disease and primary IgA nephropathy.\n\n\nConsent\n\nInformed written consent for publication of clinical details was obtained from the patient.", "appendix": "Author contributions\n\n\n\nAishwarya Damodaran, Georgi Abraham, Sanjeev Nair, Anand Yuvaraj: the nephrology team headed by Dr. Georgi Abraham took care of the patient.\n\nAnusha Rohit is a clinical microbiologist who helped make the diagnosis. All authors contributed to writing the manuscript and agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nCalhoun JH, Manring MM, Shirtliff M: Osteomyelitis of the long bones. Semin Plast Surg. 2009; 23(2): 59–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarlos GN, Kesler AK, Coleman JJ, et al.: Aggressive surgical management of sternoclavicular joint infections. J Thorac Cardiovasc Surg. 1997; 113(2): 242–247. PubMed Abstract \| Publisher Full Text\n\nPiazza C, Magnoni L, Nicolai P: Clavicular osteomyelitis: a rare complication after surgery for head and neck cancer. Eur Arch Otorhinolaryngol. 2006; 263(7): 653–6. PubMed Abstract \| Publisher Full Text\n\nJudich A, Haik J, Rosin D, et al.: Osteomyelitis of the clavicle after subclavian vein catheterization. JPEN J Parenter Enteral Nutr. 1998; 22(4): 245–246. PubMed Abstract \| Publisher Full Text\n\nBalakrishnan C, Vashi C, Jackson O, et al.: Post-traumatic osteomyelitis of the clavicle: A case report and review of literature. Can J Plast Surg. 2008; 16(2): 89–91. PubMed Abstract \| Free Full Text\n\nTice AD, Hoaglund PA, Shoultz DA: Risk factors and treatment outcomes in osteomyelitis. J Antimicrob Chemother. 2003; 51(5): 1261–1268. PubMed Abstract \| Publisher Full Text\n\nMuñoz-Fernández S, Maciá MA, Pantoja L, et al.: Osteoarticular infection in intravenous drug abusers: influence of HIV infection and differences with non drug abusers. Ann Rheum Dis. 1993; 52(8): 570–574. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDonadio JV, Grande JP: IgA nephropathy. N Eng J Med. 2002; 347(10): 738–748. PubMed Abstract \| Publisher Full Text\n\nTevlin MT, Wall BM, Cooke CR: Reversible renal failure due to IgA nephropathy associated with osteomyelitis. Am J Kidney Dis. 1992; 20(2): 185–8. PubMed Abstract \| Publisher Full Text" }	[ { "id": "6662", "date": "12 Nov 2014", "name": "K V Dakshinamurthy", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting and informative article. It however needs clarification regarding the statement that this patient is non-immunosuppressed. This patient is a patient of chronic kidney disease, which by itself is an immunosuppressed state. He received prednisolone at a dose of 50 mg per day after the diagnosis of IgA nephropathy. The duration of therapy was not mentioned. Prednisolone therapy produces a state of immunosuppression. He presented again 7 months later with swelling pain and erythema of the right clavicle that had been present for 2 months. Therefore the infection occurred in an immunosuppressed individual.", "responses": [] }, { "id": "7007", "date": "11 Dec 2014", "name": "Kashi Nath Prasad", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral commentsOsteomyelitis of right clavicle in an imuunocompetent individual is a rare entity that too by an opportunistic bacterium like Pseudomonas aeruginosa. Here the authors report osteomyelitis of medial end of clavicle caused by P. aeruginosa in an immunocompetent individual with IgA nephropathy. The case is interesting and it highlights the importance of prompt diagnosis of such cases and initiation of appropriate antibiotics. Specific commentsIt is not clear if the patient had any history of trauma or any invasive procedure on clavicle? Ultra sound guided aspiration was done in another hospital that did not yield any bacterial growth. Could infection by P. aeruginosa be iatrogenic because of earlier aspiration procedure? P. aeruginosa was found sensitive to cefoperazone and sulbactum but what about the sensitivity to other antibiotics. Authors should provide the antibiotic profile of the isolate. Could it be possible that IgA nephropathy and marked renal failure led to immunosuppressed state precipitating osteomyelitis?", "responses": [] } ]	1	https://f1000research.com/articles/3-268
https://f1000research.com/articles/3-243/v1	14 Oct 14	{ "type": "Observation Article", "title": "Plasmodium falciparum infection rates for some Anopheles spp. from Guinea-Bissau, West Africa", "authors": [ "Michelle R. Sanford", "Anthony J. Cornel", "Catelyn C. Nieman", "Joao Dinis", "Clare D. Marsden", "Allison M. Weakley", "Sarah Han", "Amabelia Rodrigues", "Gregory C. Lanzaro", "Yoosook Lee", "Michelle R. Sanford", "Anthony J. Cornel", "Catelyn C. Nieman", "Joao Dinis", "Clare D. Marsden", "Allison M. Weakley", "Sarah Han", "Amabelia Rodrigues", "Gregory C. Lanzaro" ], "abstract": "Presence of Plasmodium falciparum circumsporozoite protein (CSP) was detected by enzyme linked immunosorbent assay (ELISA) in a sample of Anopheles gambiae s.s., A. melas and A. pharoensis collected in Guinea-Bissau during October and November 2009. The percentage of P. falciparum infected samples (10.2% overall) was comparable to earlier studies from other sites in Guinea-Bissau (9.6-12.4%). The majority of the specimens collected were identified as A. gambiae which had an individual infection rate of 12.6 % across collection sites. A small number of specimens of A. coluzzii, A. coluzzii x A. gambiae hybrids, A. melas and A. pharoensis were collected and had infection rates of 4.3%, 4.1%, 11.1% and 33.3% respectively. Despite being present in low numbers in indoor collections, the exophilic feeding behaviors of A. melas (N=18) and A. pharoensis (N=6) and high infection rates observed in this survey suggest falciparum-malaria transmission potential outside of the protection of bed nets.", "keywords": [ "Malaria is among the leading causes of childhood mortality in Guinea-Bissau", "comprising 18% of mortality of children less than five years of age as of 2010 (WHO", "2010). However", "the human malaria incidence rate in Guinea Bissau varies considerably from year to year with a general decrease in recent years to about 3 children (<5 yrs of age) per thousand in some locations (Ursing et al.", "2014). Plasmodium falciparum predominates", "causing 98% cases", "followed by a few cases of Plasmodium malaria and Plasmodium ovale. Mixed infections of P. malariae", "and to a lesser extent P. ovale", "have been recorded but appear to be rare and highly variable in both Guinea-Bissau (Snounou et al.", "1993) and neighboring Senegal (Fontenille et al.", "1997a", "Fontenille et al. 1997b)." ], "content": "Introduction\n\nMalaria is among the leading causes of childhood mortality in Guinea-Bissau, comprising 18% of mortality of children less than five years of age as of 2010 (WHO, 2010). However, the human malaria incidence rate in Guinea Bissau varies considerably from year to year with a general decrease in recent years to about 3 children (<5 yrs of age) per thousand in some locations (Ursing et al., 2014). Plasmodium falciparum predominates, causing 98% cases, followed by a few cases of Plasmodium malaria and Plasmodium ovale. Mixed infections of P. malariae, and to a lesser extent P. ovale, have been recorded but appear to be rare and highly variable in both Guinea-Bissau (Snounou et al., 1993) and neighboring Senegal (Fontenille et al., 1997a; Fontenille et al. 1997b).\n\nLimited research has been conducted on the vectors and malaria parasite infection rates in Guinea-Bissau populations of Anopheles species in general and there is no data on comparative infection rates between A. gambiae and A. coluzzii and members of the A. gambiae complex. Variability is also high among the Anopheles spp. implicated as vectors in this region of West Africa in terms of both their temporal population dynamics as well as species composition among study sites (Carnevale et al., 2010; Fontenille et al., 1997a; Jaenson et al., 1994; Snounou et al., 1993).\n\nHere we present much needed data on P. falciparum infection of Anopheles spp. specimens collected from inside and around associated human habitations at eight sites in Guinea-Bissau (Table 1).\n\nNumbers (#) indicate site locations on the map of Guinea-Bissau in Figure 1. All mosquitoes were collected indoors with a single exception; samples in Ponta Anabaca were opportunistically collected outside.\n\n\nMethod\n\nMosquitoes were collected by mouth aspiration from both the island and inland areas of Guinea-Bissau (Figure 1) in 2009 between October and November, which corresponds with the time of year previously observed to have the highest infection rate in Anopheles species (Jaenson et al., 1994). The mosquito was dissected and the head and thorax were preserved in 100% ethanol for subsequent ELISA. Genomic DNA was extracted using a DNeasy extraction kit (Qiagen). Species determination of mosquitoes from the A. gambiae complex were made with the combination of species diagnostic assays (Fanello et al., 2002; Favia et al., 2001; Santolamazza et al., 2008; Scott et al., 1993) and a divergence island SNP (DIS) genotyping assay (Lee et al., 2014) while other species were identified by morphology.\n\n1: Canjufa (12.43N, 14.13W), 2: Bambadinca (12.02N, 14.86W), 3: Antula (11.91N, 15.58W), 4: Prabis (11.80N, 15.74W), 5: Abu (11.46N, 15.91W), 6: Brus (11.23N, 15.88W), 7: Ponta Anabaca (11.18N, 16.14W) and 8: Eticoga (11.16N, 16.14W).\n\nFor the Scott PCR (Scott et al., 1993) and the Fanello RFLP (Fanello et al., 2002), we used four primers (UN [5'-GTG TGG CCC TTC CTC GAT GT-3'], GA [5'-CTG GTT TGG TCG GCA CGT TT-3'], ME [5'-TGA CCA ACC CAC TCC CTT GA-3'] and AR [5'-AAG TGT CCT TCT CCA TCC TA-3']). We excluded QD primer (Scott et al., 1993) because our study site is well outside of the geographic range of this species (East Africa). A 25 µL PCR reaction containing 1X GeneAmp PCR Buffer (Applied Biosystems), 1mM MgCl2, 0.2mM of each dNTP, 0.12 µM of each primer and 0.05U AmpliTaq DNA polymerase (Applied Biosystems) was carried out for each individual. Scott PCR products were digested using Hha1 enzyme (New England Biosystems) following the protocol stated in (Fanello et al., 2002). Thermocycler conditions were 95°C for 5 min followed by thirty-five cycles of 95°C for 45 s, 50°C for 30 s and 72°C for 45 s, with a final elongation at 72°C for 7 min, and a 4°C hold.\n\nFor the Favia PCR (Favia et al., 2001), we used four primers (R5 [5'-GCC AAT CCG AGC TGA TAG CGC-3'], R3 [5'-CGA ATT CTA GGG AGC TCC AG-3'], Mopint [5'-GCC CCT TCC TCG ATG GCA T-3'] and B/S int [5'-ACC AAG ATG GTT CGT TGC-3']. A 25 µL PCR reaction containing 1X PCR Buffer (Applied Biosystems), 1.5mM MgCl2, 0.2mM of each dNTP, 0.2 µM of primer R5, 0.2 µM of primer R3, 0.16 µM of primer Mopint, 0.1 µM of primer B/S int and 0.02U DNA polymerase AmpliTaq (Applied Biosystems) was carried out for each individual. Thermocycler conditions were 95°C for 5 min followed by thirty-five cycles of 95°C for 30 s, 64°C for 30 s and 72°C for 30 s, with a final elongation at 72°C for 7 min, and a 4°C hold.\n\nFor the SINEX PCR (Santolamazza et al., 2008), we used S200 X6.1 forward [5'-TCG CCT TAG ACC TTG CGT TA-3'] and reverse [5'-CGC TTC AAG AAT TCG AGA TAC-3'] primers. A 25 µL PCR reaction containing 1X PCR Buffer (Applied Biosystems), 2mM MgCl2, 0.4mM of each dNTP, 0.2 µM of each primer and 0.1U DNA polymerase AmpliTaq (Applied Biosystems) was carried out for each individual. Thermocycler conditions were 95°C for 5 min followed by thirty-five cycles of 95°C for 30 s, 60°C for 30 s and 72°C for 30 s, with a final elongation at 72°C for 10 min, and a 4°C hold.\n\nThe resulting PCR products were analyzed on a Qiaxcel capillary electrophoresis instrument (Qiagen) using a DNA Screening Cartridge (Qiagen).\n\nFor DIS genotyping, we used Sequenom iPLEX Gold Genotyping Reagent Set (Catalog number: Sequenom 10158) and ran on MassArray (Sequenom) mass spectrometer at UC Davis Veterinary Genetics Laboratory. A mosquito was considered a hybrid if at least 5 out of 7 DIS on the X chromosome were in a heterozygous state.\n\nP. falciparum infection was determined by enzyme linked immunosorbent assay (ELISA) of circumsporozoite protein (CSP) (Burkot et al., 1984; Wirtz et al., 1987) from the head and thorax of mosquito specimens in an attempt to capture the parts of the mosquito that would indicate they were infective mosquitoes. All chemicals except for substrate solutions (Item 5 on page 5 of the supplemental ELSA protocol document) were ordered from Sigma-Aldrich. Monoclonal antibodies (capture and conjugate) were obtained from Kirkegaard & Perry Laboratories. P. falciparum sporozoite protein for positive control was ordered from the Centers for Disease Control and Prevention (CDC). We followed the Sporozoite ELISA directions provided by the CDC (Sep, 2009 version) with a few modifications (see supplemental document for the modified ELISA protocol). Samples were considered positive if absorbance values were three or more standard deviations from the negative control samples (99% CI) on each ELISA plate (Beier et al., 1987; De Arruda et al., 2004).\n\nThe results of the ELISA were analyzed for both CSP concentration, adjusted for plate-to-plate variation, with an analysis of variance and for a binary outcome using a χ2 test implemented in SPSS 16.0 (SPSS, 2007). The data were analyzed for differences between species and among collection sites, using G-test implemented in Deducer library under R software (http://www.r-project.org/). Species and P. falciparum infection state and CSP concentration for each individual is provided in Dataset 1.\n\n\nResults & discussion\n\n\n\nFour species were collected during sampling; A. coluzzii, A. gambiae, A. melas, A. pharoensis and A. coluzzi x A. gambiae hybrids were observed. All mosquitoes were collected indoors with a single exception; samples in Ponta Anabaca were opportunistically collected outside of a human habitation while apparently host-seeking immediately after sunset at about 18:00 hr, which is earlier than reported observations for members of the A. gambiae complex in The Gambia (West Africa) (Lindsay et al., 1989; Snow et al., 1988). All species were collected at multiple sites except A. pharoensis, which was only collected at the more inland site of Bambadinca. A. pharoensis is not generally considered a significant vector in West Africa but the distribution observed in this study matches the previously observed pattern in Senegal (Carrara et al., 1990). Anopheles arabiensis was absent from collections.\n\nNo significant differences were observed for CSP concentration or in the analysis of positive samples with χ2. This is probably due to the variation in the distribution of vector species and P. falciparum in the environment at the time of sampling. Table 1 presents CSP rate data and the total number of each individual species collected at each site.\n\nThe percentage of P. falciparum positive samples from members of the A. gambiae species complex observed in this study (overall 10.2%) were similar to earlier studies in other regions in Guinea-Bissau (12.0% (Snounou et al., 1993) and 9.6–12.4% (Jaenson et al., 1994)). The overall CSP positive rate for A. gambiae was 12.6% and 11.1% for A. melas. Previously published CSP positive rates for A. gambiae s.s. (=A. gambiae and A. coluzzii) range between 2.24% in Guinea (Carnevale et al., 2010) to 9.6% in Guinea-Bissau (Jaenson et al., 1994). Earlier studies when individual species within the A. gambiae complex were not identified, infection rate of A. gambiae s.l. ranged from as high of 17.73% in the eastern regions of The Gambia (Thomson et al., 1994) to 12% in Guinea-Bissau (Jaenson et al., 1994; Snounou et al., 1993). The CSP positive rate was significantly higher in A. gambiae (12.6%) than A. coluzzii (4.3%) (Wilcoxon rank sum test P-value=0.0384). This is consistent with the earlier study in Senegal (Ndiath et al., 2011) but differs from a recent survey conducted in Mali (Fryxell et al., 2012). The study site in Senegal located in the village of Dielmo (13°43'N, 16°24'W) (Ndiath et al., 2012)) was geographically closer (200km) than Mali sites (>800km) to our collection sites in Guinea-Bissau. The Senegal study site at Dielmo and nine of our study sites were proximal (<50km) to the Atlantic Ocean, while Mali is a land-locked country at least 500km away from the Atlantic Ocean. Therefore, the discrepancy among studies may be due to climatic and environmental pressure on the different genetic backgrounds of A. gambiae observed in this area of West Africa (Lee et al., 2013). More robust sampling over a larger number of collection sites would help in confirming this trend.\n\nIn this study, a few A. pharoensis (N=6) were collected, half of which were CSP positive. Other studies in this region of West Africa have found that A. funestus and A. arabiensis may also be important vector species at different times in nearby Senegal (Fontenille et al., 1997a; Fontenille et al., 1997b). A. arabiensis was not collected in our study while a small number (N<10) of A. funestus were observed but not collected.\n\nRecent studies on the prevalence of malaria parasites in humans have suggested that infection rates in Guinea-Bissau may be in decline due to widespread use of effective treatment and insecticide treated bed nets (ITNs and long lasting insecticide treated bed nets, LLINs) by the most high-risk groups (Rodrigues et al., 2008; Ursing et al., 2014). The malaria parasite life cycle is complicated and may not directly relate to the prevalence of human cases but it is possible that the lack of data during periods of political unrest has concealed a more stochastic pattern than was previously observed in Guinea-Bissau (Ursing et al., 2014).\n\nOutdoor mosquito collection was not the focus of this survey and was only made at Ponta Anabaca Hotel grounds when we fortuitously noted mosquitoes biting. Consequently no general comments about the degree of exophily of A. gambiae in Guinea-Bissau can be made. However, evidence of exophily by the major malaria vector A. gambiae in this study and by others in West Africa (Reddy et al., 2011; Tchouassi et al., 2012) raises the concern of the long term effectiveness of Indoor Residual Spraying (IRS) and Long lasting Insecticide-treated Nets (LLINs) in reducing outdoor transmission of malaria especially before bedtime and by people sleeping outdoors. The relatively high infection rate of 11.1% of A. melas in Guinea-Bissau together with its tendencies to be both endophilic and exophilic and have a high human blood index (Sharp et al., 2007; Tuno et al., 2010) make the species a significant vector, which may also be hard to control by reliance on ITNs and LLINs.\n\nThe high CSP rate of 33.3 % in the 4 indoor collected A. pharoensis might implicate a significant role in malaria transmission in drier inland Guinea Bissau, however this should be viewed with caution due the small sample size. Very low infection rates and absence of malarial parasites, traditionally found in West and Central African populations of A. pharoensis has always led to the conclusion that this mosquito plays little role in malaria transmission despite its anthropophilic habits and that it can be easily experimentally infected (DeMeillon, 1947; Ndiath et al., 2012; Tchouassi et al., 2012). In drier Sahel regions of Africa where the major vectors of malaria are absent or very rare and irrigated rice and other crop lands are increasing, A. pharoensis is considered more important at maintaining low levels of malaria (Kerah-Hinzoumbe et al., 2009; Kibret et al., 2010).\n\n\nData availability\n\nfigshare: ELISA results identifying Plasmodium falciparum infection status in Anopheles spp. collected in Guinea-Bissau. doi: 10.6084/m9.figshare.1200058 (Sanford et al., 2014).", "appendix": "Author contributions\n\n\n\nYL, GCL and AJC conceived the study, designed experiments and conducted field collections. JD conducted field collection. AR provided logistical support and coordination for field collection in Guinea-Bissau. CCN, CDM, AMW and SH conducted DNA extraction, ELISA and PCR. MRS performed data analysis and wrote manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe authors also acknowledge financial support from NIH grants: 5R21AI062929 and 5T32AI074550.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank Parker Goodell and Mike Kim for their assistance in DNA extraction, molecular form determination and ELISA processing. We thank Julia Malvick at the Veterinary Genetics Laboratory of UC Davis School of Veterinary Medicine for assistance in processing iPLEX SNP genotyping assay.\n\n\nSupplementary materials\n\nSporozoite ELISA Directions. Click here to access the supplement.\n\n\nReferences\n\nBeier JC, Perkins PV, Wirtz RA, et al.: Field evaluation of an enzyme-linked immunosorbent assay (ELISA) for Plasmodium falciparum sporozoite detection in anopheline mosquitoes from Kenya. Am J Trop Med Hyg. 1987; 36(3): 459–468. PubMed Abstract\n\nBurkot TR, Williams JL, Schneider I: Identification of Plasmodium falciparum-infected mosquitoes by a double antibody enzyme-linked immunosorbent assay. Am J Trop Med Hyg. 1984; 33(5): 783–788. PubMed Abstract\n\nCarnevale P, Toto JC, Guibert P, et al.: Entomological survey and report of a knockdown resistance mutation in the malaria vector Anopheles gambiae from the Republic of Guinea. Trans R Soc Trop Med Hyg. 2010; 104(7): 484–489. PubMed Abstract \| Publisher Full Text\n\nCarrara GC, Petrarca V, Niang M, et al.: Anopheles pharoensis and transmission of Plasmodium falciparum in the Senegal River delta, West Africa. Med Vet Entomol. 1990; 4(4): 421–424. 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PubMed Abstract \| Publisher Full Text\n\nUrsing J, Rombo L, Rodrigues A, et al.: Malaria transmission in Bissau, Guinea-Bissau between 1995 and 2012: malaria resurgence did not negatively affect mortality. PLoS One. 2014; 9(7): e101167. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWHO: Guinea-Bissau Factsheets of Health Statistics 2010, pp. World Health Organization. 2010. Reference Source\n\nWirtz RA, Zavala F, Charoenvit Y, et al.: Comparative testing of monoclonal antibodies against Plasmodium falciparum sporozoites for ELISA development. Bull World Health Organ. 1987; 65(1): 39–45. PubMed Abstract \| Free Full Text" }	[ { "id": "6413", "date": "22 Oct 2014", "name": "Jacques Derek Charlwood", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper provides information on sporozoite rates from a relatively unstudied area the mainland and islands of Guinea Bissau. It gives the impression that it is a spin off from another study that perhaps aimed at characterizing the genetics of populations of Anopheles gambiae from the mainland and the islands, perhaps for future genetic control efforts. Given the widespread use of mosquito bednets rates are exceptionally high and not, apparently different from rates recorded earlier. The authors do make the comment that this might be the result of civil strife in Guinea-Bissau but whatever the cause this is disquieting and implies that the gains in reduction of malaria are going to be at best temporary. The data are presented without confidence intervals but these should be added. Given the relatively small numbers involved either adjusted Wald confidence intervals (that can easily be calculated using the site www.measuringu.com/wald.htm or a routine in R) can, I think, be used. (But since I am signing this review everyone should know that my statistical abilities are limited! The kind of collection undertaken needs to be explained in more detail. Were the mosquitoes collected resting or were they landing collections? I do not really want to be the person raising this issue but something on ethics should be included somewhere. (My own thoughts on ethics in general is that if the rule of ‘first do no harm and second maybe do some good’ is followed then a study – that may include even ad hoc landing collections – is not unethical.) This is especially important if the collections were landing collections. To avoid possible misunderstanding, the sentence ‘Four species were collected during sampling; A. coluzzii, A. gambiae, A. melas, A. pharoensis and A. coluzzi x A. gambiae hybrids were observed ‘ should be rewritten (since it could be misinterpreted) perhaps as two sentences: ‘Four species, A. coluzzii, A. gambiae, A. melas, A. pharoensis were collected during sampling. A number of A. coluzzi x A. gambiae hybrids were also collected’ There are a number of small errors in the paper that need to be rectified. For example in the last paragraph they state ‘33% of the 4 Anopheles pharoensis collected indoors when they either mean 33% of the six Anopheles pharoensis collected or 50% of the four collected indoors. With regard to this species it may be worth pointing out that in Mozambique none of the 4390 tested were positive for sporozoites (Charlwood et al., 2013) but at the same time in Ghana, (Dzodzomenyo et al., 1999) found that two of three specimens of An. pharoensis examined were infected (with Bancrotian filariasis) and one of these was infectious. Given the possibility of false positives among primarily zoophilic anophelines (that may also include An. melas) and given that the authors have access to a sophisticated laboratory it is a shame that they did not run a PCR on the sporozoite positive specimens to ensure that they were indeed human malarias.", "responses": [ { "c_id": "1058", "date": "31 Oct 2014", "name": "Yoosook Lee", "role": "Reader Comment", "response": "Thank you very much for your review. Revisions to the manuscript will be made as suggested.WIth respect to you comment about CI, the data presented in the table are not replicated but a simple calculation of the percent of the total collection that were infected. We did not replicate within time or locations. Therefore there is no variation to report in the table. About the concern related to outdoor mosquito collection, our only outdoor collection is from Ponta Anabaca where the hotel was located where we stayed. While we were processing our sample collections in the early evening at the hotel, we (Drs. Cornel, Lee and Lanzaro) were harassed by mosquitoes, which we identified as Anophelines by morphology. Although this was unplanned ad hoc landing collections, we were all taking anti-malaria prophylaxis at the time. About the concern you raised about false positive of ELISA, this is a possibility for any test of parasite detection. We specifically chose to use the P. falciparum circumsporozoite protein sensitive ELISA test on the parts of the mosquito most likely to contain the infective stages (head and thorax) to minimize false positives in the results. Unfortunately the ELISA test was destructive and these specific parts of the mosquito have long been discarded. The DNA from the remainder of each mosquito has been archived but this contains the abdomen which presents another potential source of false positives as the gut contents may contain material that may never progress to rendering the mosquito infective.Another confounding factor associated with using this type of testing is that it needs to be conducted more rapidly than the DNA extraction from the rest of the mosquito and the ELISA tests were often performed before the mosquito identification via PCR could be conducted. Such that an unusual result such as the one observed here would not have been detected until the ELISA samples had been discarded. It would definitely be beneficial to keep this in mind for future work in this area." } ] }, { "id": "6412", "date": "23 Oct 2014", "name": "Guido Favia", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis “observation article” is very well written in a format that is accessible to both general and specialist audience. It describes some novel observations about malaria infection rates in different vector species in Guinea-Bissau. In particular it reports the some how unexpected high infection rates in Anopheles melas and A. pharoensis, thus suggesting Plasmodium falciparum-malaria transmission potential outside of protection (i.e. bed nets).Details about the circumstances of the finding and evidence of the observation are properly provided. The manuscript appropriately cites relevant bibliography in the field. Figures and tables are informative and helpful. Methods section is well organized and nicely descriptive.As observational article it looks perfectly adequate to the journal purpose.I have only a very minor concern: in the Introduction (line 8) Plasmodium malaria should be re-written as Plasmodium malariae.", "responses": [ { "c_id": "1057", "date": "31 Oct 2014", "name": "Yoosook Lee", "role": "Reader Comment", "response": "Thank you very much for your review. Revisions to the manuscript were made as suggested." } ] } ]	1	https://f1000research.com/articles/3-243
https://f1000research.com/articles/3-218/v1	11 Sep 14	{ "type": "Research Article", "title": "Monitoring drug promiscuity over time", "authors": [ "Ye Hu", "Jürgen Bajorath", "Ye Hu" ], "abstract": "Drug promiscuity and polypharmacology are much discussed topics in pharmaceutical research. Experimentally, promiscuity can be studied by profiling of compounds on arrays of targets. Computationally, promiscuity rates can be estimated by mining of compound activity data. In this study, we have assessed drug promiscuity over time by systematically collecting activity records for approved drugs. For 518 diverse drugs, promiscuity rates were determined over different time intervals. Significant differences between the number of reported drug targets and the promiscuity rates derived from activity records were frequently observed. On the basis of high-confidence activity data, an increase in average promiscuity rates from 1.5 to 3.2 targets per drug was detected between 2000 and 2014. These promiscuity rates are lower than often assumed. When the stringency of data selection criteria was reduced in subsequent steps, non-realistic increases in promiscuity rates from ~6 targets per drug in 2000 to more than 28 targets were obtained. Hence, estimates of drug promiscuity significantly differ depending on the stringency with which target annotations and activity data are considered.", "keywords": [ "Drug promiscuity", "pharmacology", "activity data" ], "content": "Introduction\n\nPromiscuous compounds specifically interact with multiple biological targets1. As such, they are distinct from compounds that exhibit assay liabilities or engage in various non-specific interactions. Compound promiscuity is often functionally relevant and represents the molecular origin of polypharmacology2, a concept that experiences increasing interest in drug discovery. Drugs are often, but not always, found to act on multiple targets and modulate multiple cellular pathways and/or signaling cascades. Such effects might often substantially contribute to therapeutic efficacy, for example, in cancer treatment3. The potentially far reaching consequences of drug polypharmacology for therapy, the frequency of these effects, and likely pros and cons are just beginning to be understood.\n\nExperimentally, promiscuity can be assessed by profiling of compounds or drugs on arrays of biological targets1,2, although such studies might often only provide an incomplete picture of in vivo effects. The same applies to computational estimates of promiscuity. Given the increasingly large amounts of compound activity data that are becoming available, the promiscuity of drugs and bioactive compounds can be explored through data mining by systematically evaluating activity annotations1. For the assessment of compound and drug promiscuity, public databases such as ChEMBL4, the major repository of compounds and activity data from medicinal chemistry, the PubChem BioAssay database5, the major repository of screening data, and DrugBank6, which collects approved and experimental drugs, have become indispensible resources.\n\nComputational analyses reported thus far have suggested different degrees of promiscuity among bioactive compounds and drugs, dependent on the compound sources used and the methods applied. For example, drug-target network analysis has indicated that a drug might on average act on two targets7. Other computational studies have suggested that drugs might on average interact with two to seven targets depending on the target classes the drugs are active against8. In addition to varying compound sources and analysis concepts, taking activity measurement characteristics and data confidence criteria into account is also of critical importance for compound promiscuity analysis. For example, it has been shown that the increase in the number of compounds with activity against targets from different families in ChEMBL has mostly resulted from assay-dependent IC50 but not (assay-independent) Ki measurements (equilibrium constants)9. In addition, by exclusively considering high-confidence activity data, it has been found that the most promiscuous bioactive compounds interact with two to five targets from the same target family, are predominantly active in sub-µM range, and display potency differences within one or two orders of magnitude against their targets10. This represents a prevalent promiscuity profile among bioactive compounds. On the basis of high-confidence activity data, it has also been calculated that compounds from ChEMBL interact on average with one to two targets and compounds from PubChem confirmatory assays with two to three targets11. By contrast, target annotation analysis has suggested that approved drugs interact on average with close to six targets, whereas experimental drugs (including candidates in clinical trials) interact with one to two targets11. The reasons for this apparent discrepancy in target numbers between drugs at different development stages are currently unknown. As increasing amounts of activity data become available, it is likely that recently detected promiscuity rates might further increase. However, the magnitude of such increases as a consequence of data incompleteness12 is difficult to predict, especially considering the low promiscuity rates that can currently be confirmed on the basis of high-confidence data1,11.\n\nIn this study, we further extend the computational analysis of promiscuity by evaluating the progression of drug promiscuity rates over time, which required a systematic assessment of activity records with release dates. Different data selection criteria were applied and the calculated promiscuity rates were compared to available drug target annotations. Small to moderate increases in drug promiscuity over time were detected when high-confidence activity data were considered. Lowering the stringency of data selection criteria led to unrealistic estimates of promiscuity rates and their progression.\n\n\nMaterials and methods\n\nFrom ChEMBL (release 18)4, compounds with direct interactions (i.e., assay relationship type “D”) with human targets at the highest confidence level (i.e., assay confidence score 9) were collected. The two ChEMBL parameters ‘assay relationship type’ and ‘assay confidence score’ qualify and quantify the level of confidence that the activity against a given target is evaluated in a relevant assay system, respectively. Accordingly, type “D” and score 9 represent the highest level of confidence for activity data. In addition, two types of activity measurements were considered including assay-independent equilibrium constants (Ki values) and assay-dependent IC50 values. To ensure a high level of data integrity, only compounds with explicitly defined Ki or IC50 values were selected. Hence, approximate measurements such as “>”, “<”, and “~” were disregarded. Compounds with multiple Ki or IC50 measurements for the same target were retained if all these values fell within the same order. Otherwise, the target activity was omitted from further consideration. Structures of all qualifying bioactive compounds were standardized using the Molecular Operating Environment (MOE)13 and transformed into canonical SMILES strings14. The so assembled compound set exclusively utilized high-confidence activity data (high-confidence data set).\n\nApproved small molecule drugs with available structure and activity information were collected from the latest release of DrugBank (version 4.1)6. To synchronize the activity analysis in ChEMBL and DrugBank, all reported ‘drug action’ targets, metabolizing enzymes, transporters, and carriers were assembled for approved drugs. In some instances, drug target activity might refer to a group of related proteins. For example, atomoxetine was annotated with N-methyl-D-aspartate (NMDA) receptor including seven subtypes. Accordingly, seven UniProt15 accession IDs (UniProtIDs) were associated with NMDA receptor. Thus, the maximal number of target annotations was collected for approved drugs on the basis of UniProtIDs. Drug structures were also standardized using MOE and transformed into canonical SMILES strings.\n\nMost compound activity data in ChEMBL are extracted from medicinal chemistry literature and patent sources4. Therefore, the release dates of activity data are frequently recorded in this database. However, DrugBank does not report dates for individual target annotations. To systematically monitor drug promiscuity over time, all approved drugs from DrugBank were mapped to ChEMBL by comparing canonical SMILES strings. If a drug (D) and a bioactive compound (B) shared the same SMILES string, a match was obtained. It should be noted that the name of a drug in DrugBank and ChEMBL might differ (i.e., matching by drug/compound name is not reliable). For each match, activity data release dates of compound B were recorded and assigned to drug D. Each activity record represented a target annotation (the terms target activity and target annotation are synonymously used). For instance, if compound B was reported to be active against target I in 2001, target II in 2005, and target III in 2009, the cumulative activity records for drug D consisted of target I in 2001, targets I and II in 2005, and targets I, II, and III in 2009. Thus, the promiscuity rate of D increased over time from 1 to 3. All activity records were organized into 14 time intervals, as illustrated in Figure 1. All activity records reported before 2000 were assigned to 2000, the starting point of our analysis, and all activity data released after 2012 were assigned to the last period “>2012”. For each time interval, the cumulative activity profile was recorded. Hence, changes in the promiscuity rate of a drug were successively determined over the years. Cumulative activity profiles were compared to target annotations available in DrugBank.\n\nThe organization of the activity records for a drug over different years is schematically illustrated. Drug D and a bioactive compound B share the same SMILES string (D is mapped to B). The activity records of compound B are extracted from ChEMBL. B is reported to be active against target I in 2001, II in 2005, and III in 2009. These activity records are then assigned to drug D and organized into 14 time intervals (12 of which represent individual years, except 2000 (see text) and >2012). For each interval, a cumulative activity profile is generated for D and recorded. The total number of activity annotations is given in red.\n\nIn order to investigate the effect of activity data confidence levels on drug promiscuity, two data sets with lower confidence were assembled from ChEMBL (release 18). For the generation of low-confidence data sets, two criteria that influence the compound data integrity, i.e., the confidence level of activity and the type of activity measurements were disregarded in subsequent steps. In low-confidence set 1, the criterion of activity measurement type was not considered. Hence, in addition to Ki and IC50 values, all other potency annotations were equally considered (including “%max”, “Efficacy”, “EC50”, “Kd”, and “Residual Activity”) for all compounds with ‘direct interactions’ with human targets and assay confidence score 9. In low-confidence set 2, the confidence level of activity (assay relationship type and assay confidence score) was not considered, in addition to the type of activity measurements. Therefore, the stringency of activity data and compound selection decreased from the high-confidence set over low-confidence set 1 to low-confidence set 2.\n\nProgression of drug promiscuity over time was systematically evaluated on the basis of all three data sets.\n\n\nResults and discussion\n\nOn the basis of the selection criteria described above, a total of 143,424 bioactive compounds with high-confidence activity data were obtained from ChEMBL. These compounds were active against 1376 different targets and yielded 219,602 compound-target interactions, as reported in Table 1. Furthermore, from DrugBank 4.1, 1429 approved drugs were obtained that were annotated with 1657 target proteins corresponding to 10,679 drug-target interactions (Table 1). Thus, there were nearly 100 times more bioactive compounds than approved drugs. However, with 1657 targets, drugs covered a larger target space than bioactive compounds (1376 targets). On average, a bioactive compound was active against 1.5 targets, whereas an approved drug was annotated with 7.5 targets. Compared to a recent analysis of promiscuity rates11, which also included a previous release of DrugBank, the average promiscuity rate of approved drugs further increased from 5.9 to 7.5, while the degree of promiscuity among bioactive compounds remained essentially constant.\n\nFor DrugBank 4.1 (drugs) and ChEMBL release 18 (compounds), the number of drugs/compounds, targets the drugs/compounds were active against, and the total number of interactions is reported.\n\nTo monitor drug promiscuity over time, all approved drugs were mapped to bioactive compounds in ChEMBL for which release dates of activity records were reported (as detailed in the Methods section). For 518 of the 1429 approved drugs taken from DrugBank, high-confidence activity data released over different years were found in ChEMBL. These 518 drugs provided the basis for our time-dependent promiscuity analysis.\n\nFor the 518 qualifying drugs, we first compared their target annotations in DrugBank and the total number of targets derived from high-confidence activity records in ChEMBL. As reported in Figure 2a, most of the drugs had different numbers of targets in the two databases. Only 32 drugs (~6%) were found to have the same number of target annotations in DrugBank and ChEMBL. The total number of target annotations of a drug represented its promiscuity rate. A total of 439 drugs had higher promiscuity rates in DrugBank than in ChEMBL. Opposite observations were only made for 47 drugs. On average, the 518 drugs were annotated with ~10.1 targets in DrugBank and ~3.2 targets derived from high-confidence ChEMBL activity records. Hence, promiscuity rates in DrugBank were much higher than in ChEMBL. Exemplary drugs having the same or different degrees of promiscuity in DrugBank and ChEMBL are shown in Figure 2b–Figure 2d.\n\n(a) For 518 qualifying drugs, the number of targets reported in DrugBank and the number of high-confidence target-dependent activity annotations in ChEMBL are compared in a scatter plot. Each dot represents a drug. The diagonal (indicating perfect correlation) is drawn in red. In (b), (c), and (d), exemplary drugs are shown that had the same number of targets in DrugBank and ChEMBL (i.e., the same promiscuity rate), a higher promiscuity rate in DrugBank, and a higher rate in ChEMBL, respectively. For each drug in (b), the number of targets is given. For each drug in (c) and (d), the numbers of targets reported in DrugBank and ChEMBL are compared. For example, “10 \| 6” indicates that the drug was annotated with 10 targets in DrugBank and with six in ChEMBL.\n\nDifferences in promiscuity rates were quantified, as reported in Figure 3a. Among the 486 drugs (~94%) with varying degrees of promiscuity in DrugBank and ChEMBL, 48 and 58 drugs differed by one and two targets, respectively. By contrast, the promiscuity rates of nearly half of the drugs (247; ~48%) varied by more than five targets. Moreover, for the 10 drugs shown in Figure 3b, the promiscuity rates differed by more than 30 targets, which reflected a particularly high degree of data inconsistency. All of these drugs were annotated with many more targets in DrugBank than targets derived from high-confidence activity records in ChEMBL. The extreme case was olanzapine the promiscuity rate of which differed by 47 targets between the two databases.\n\n(a) Reported is the distribution of promiscuity differences (∆Activities) between DrugBank and ChEMBL for 518 drugs. (b) Shown are 10 drugs with the largest difference in promiscuity (∆Activities > 30). Target annotations are represented according to Figure 2c and Figure 2d.\n\nIn addition to comparing the number of target annotations, the activity profiles of drugs were further examined to determine the consistency of the annotations. As reported in Figure 4, 175 drugs (~34%) had non-overlapping sets of targets in these two databases, which was another surprising finding. The remaining 343 drugs had overlapping yet distinct target sets. However, the majority of these drugs shared only one or two targets, reflecting substantial discrepancies between target annotations.\n\nThe activity profiles of 518 drugs in DrugBank and ChEMBL are compared. Reported is the number of drugs sharing increasing numbers of activities (#Common activities) in the two databases.\n\nFor the study of changes in drug promiscuity over time, accessing original activity records and their release dates was an essential requirement, as rationalized above. Such information is not available in DrugBank.\n\nNext, we organized the 518 drugs on the basis of activity record release dates. Drugs were assigned to the individual time intervals in which high-confidence activity data were first published. For example, if the first activity record of a given drug was detected in 2005, the drug was assigned to the 2005 interval and traced during all subsequent years. The cumulative number of drugs in different time intervals is reported in Figure 5a. By 2000, high-confidence activity data were publicly available for 78 drugs. From 2000 to 2001, activity data became available for 26 additional drugs. The number of drugs for which qualifying activity records were available in subsequent years ranged from 20 to 64, with an average of ~34 drugs per interval. The largest increase was detected for 2007/2008. The time period for which the activity records were assembled spanned a maximum of 24 years (for captopril, from 1981 to 2005), with an average of 3.3 years per drug. Exemplary drugs for which activity records were first reported before 2000 and after 2008 are shown in Figure 5b and Figure 5c, respectively.\n\n(a) Reported is the cumulative number of drugs for which high-confidence activity data became available in different years. In (b) and (c), six exemplary approved drugs are shown for which high-confidence activity data were first recorded before 2000 or after 2008, respectively. For each drug, its name, year of first data report, and therapeutic indication are provided.\n\nFor individual time intervals, the distribution of drug promiscuity rates was determined, as reported in Figure 6a. The box plots reveal an increase in drug promiscuity rates over time, with a maximal rate of six targets per drug in 2000 and 24 targets per drug in interval >2012. However, median promiscuity rates only slightly increased from one (until 2005) to two (beginning in 2006) targets per drug. The distribution of average promiscuity rates is shown in Figure 6b, which slightly but steadily increased over time from 1.5 to 3.2 targets per drug. The larger relative increase of average than median promiscuity rates indicated that the average values were influenced by small numbers of drugs with large numbers of targets, i.e., a small subset of highly promiscuous drugs, consistent with earlier observations11. On the basis of median values, detectable increases in drug promiscuity over time were limited.\n\n(a) Box plots capture the distribution of the number of targets per drug in different years. Each box plot reports the smallest value (bottom line), lower quartile (lower boundary of the box), median (thick horizontal line), upper quartile (upper boundary of the box), and the largest value (top line). (b) Reported are average numbers of targets per drug in different years.\n\nChanges in promiscuity over time were also monitored for individual drugs. For each drug, the increase in the cumulative promiscuity rates from its first to its most recent activity records was determined (for the hypothetical example in Figure 1, the increase in promiscuity rate is 2). For the 518 drugs, increases are reported in Table 2. Surprisingly, for 282 drugs (~54%), no increase in promiscuity was detected on the basis of high-confidence activity records. Exemplary drugs with constant promiscuity rates are shown in Figure 7. For the remaining 236 drugs, increasing numbers of targets were detected. However, in most cases, the increase in target numbers was limited, i.e., the promiscuity rates of 197 drugs increased by one to five targets (Table 2). There were only 14 drugs with an increase in promiscuity rates by 10 or more targets. Five drugs with largest increase in promiscuity rates are shown in Figure 8. For example, the promiscuity rate of imatinib increased from one in 2002 to 24 (>2012), with 11 new targets reported between 2008 and 2009. The drugs in Figure 8 belonged to the subset of highly promiscuous drugs that statistically influenced the calculation of average promiscuity rates, as discussed above.\n\nThe number (percentage) of drugs with increasing promiscuity rates (i.e., number of targets) is reported.\n\nShown are 12 exemplary drugs having a constant promiscuity rate on the basis of high-confidence activity data. For each drug, the year of its first activity report and the number of targets it was active against are given. For example, brimonidine was first reported to be active against a single target in 1997.\n\nFor the five drugs with largest changes in promiscuity over time, cumulative numbers of targets are reported for different years (top). For each drug, the overall difference in target annotations is given in parentheses. The structures of these drugs are shown at the bottom.\n\nDrug promiscuity across different target families was also assessed. For the 236 drugs with increases in promiscuity rates over time, their targets were assigned to families and the number of target families was determined and followed over time. Table 3 reports the number of drugs with increasing target family annotations. For the majority of drugs, the number of target families increased by one or two. For 47 drugs, the number of target families remained constant.\n\nThe number (percentage) of drugs with increasing target family promiscuity (i.e., increasing number of protein families the drug targets belong to) is reported.\n\nTwo compound sets with lower activity data confidence were also assembled from ChEMBL, as described above. The composition of these sets is summarized in Table 4. Low-confidence set 1 in which the types of activity measurements were not specified contained a total of 605,206 compounds active against 2144 targets, yielding more than 2,600,000 interactions. Low-confidence set 2 in which, in addition, the confidence level of activity was undefined consisted of a larger number of 936,924 compounds active against 3934 targets, yielding more than 6,000,000 interactions. All 518 drugs were mapped to these two low-confidence data sets. The cumulative distribution of these drugs over time is reported in Figure 9a. The number of drugs with low-confidence activity annotations in 2000 increased from 78 (high-confidence set) to 194 (low-confidence set 1) and 335 (low-confidence set 2). On average, ~26 and ~15 drugs became available during each year for low-confidence set 1 and 2, respectively.\n\nFor both low-confidence sets (see text for details), the number of compounds, targets, and interactions is reported.\n\n(a) Reported is the cumulative number of drugs in three data sets of varying confidence levels over time. (b) Shown is the distribution of average promiscuity rates for drugs in these three data sets. (c) For imatinib, the cumulative number of targets is reported for different years.\n\nFigure 9b compares the distribution of average drug promiscuity rates for the three data sets over time. In contrast to the high-confidence data set in which drug promiscuity only slightly increased over the years, the average promiscuity rates of drugs in both low-confidence sets were higher and significantly increased. In low-confidence set 2, the average promiscuity rate was 6.3 targets per drug in 2000 and further increased to 28.2 targets (>2012). Thus, by reducing the stringency of selection criteria for activity records, high average promiscuity rates were obtained. The large increases in average promiscuity rates seen in Figure 9b ultimately resulted in 18 (low-confidence set 1) or nearly 30 (set 2) targets per drug are most likely artificial in nature. The comparison reveals how the choice of different activity data selection criteria, or the lack of well-defined criteria, might bias promiscuity analysis.\n\nImatinib represented a striking example for the presence of unreliable target annotations under non-stringent data selection criteria (Figure 9c). In both low-confidence sets 1 and especially 2 dramatic increases were observed between 2005 and 2008, ultimately leading to 406 and 689 targets for imatinib, respectively (hence exceeding the total number of targets in the human kinome). By contrast, on the basis of high-confidence activity data, the final (>2012) promiscuity rate of imatinib was 24.\n\n\nConclusions\n\nThe analysis reported herein was designed to monitor drug promiscuity over time through computational data mining. It was facilitated by systematically collecting available activity records with release dates for approved drugs from the CHEMBL database. For more than 500 drugs, it was possible to assess promiscuity rates over a time course. Current promiscuity rates derived from high-confidence ChEMBL activity records are typically much lower than those calculated from target annotations available in DrugBank, which should merit further consideration. Data selection criteria for the assignment of drug targets might at least in part be responsible for the observed differences. On the basis of high-confidence activity data, an increase in the average drug promiscuity rates from only 1.5 to 3.2 targets per drug was observed. The magnitude of average promiscuity rates was influenced by a small subset of highly promiscuous drugs. Thus, increases in average drug promiscuity over time were generally small. However, they frequently involved targets from at least two families. By contrast, for low-confidence data sets, calculated promiscuity rates were much higher and dramatic increases in apparent drug promiscuity were observed over the years. From our point of view, such trends are unreliable. These observations further emphasize the need for well-defined and stringent data selection criteria for promiscuity analysis. Taken together, the findings reported herein reveal a small to moderate increase in detectable drug promiscuity over time while the volumes of compound activity data rapidly grow.\n\n\nData availability\n\nThe high-confidence and the two low-confidence drug data sets are made available in ZENODO. For each drug in each set, the ChEMBL activity records are provided for individual time intervals.\n\nZENODO: Drug activity data, doi: 10.5281/zenodo.1157616", "appendix": "Author contributions\n\n\n\nJB conceived the study, YH planned and performed the analysis, YH and JB wrote the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nHu Y, Bajorath J: Compound promiscuity: what can we learn from current data? Drug Discov Today. 2013; 18(13–14): 644–650. PubMed Abstract \| Publisher Full Text\n\nBoran AD, Iyengar R: Systems approaches to polypharmacology and drug discovery. Curr Opin Drug Discov Devel. 2010; 13(3): 297–309. PubMed Abstract \| Free Full Text\n\nKnight ZA, Lin H, Shokat KM: Targeting the cancer kinome through polypharmacology. Nat Rev Cancer. 2010; 10(2): 130–137. 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Publisher Full Text\n\nHu Y, Bajorath J: Growth of ligand-target interaction data in ChEMBL is associated with increasing and measurement-dependent compound promiscuity. J Chem Inf Model. 2012; 52(10): 2550–2558. PubMed Abstract \| Publisher Full Text\n\nHu Y, Bajorath J: Activity profile relationships between structurally similar promiscuous compounds. Eur J Med Chem. 2013; 69: 393–398. PubMed Abstract \| Publisher Full Text\n\nHu Y, Bajorath J: High-resolution view of compound promiscuity. [v2; ref status: indexed, http://f1000r.es/1ig]. F1000Res. 2013; 2: 144. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMestres J, Gregori-Puigjané E, Valverde S, et al.: Data completeness--the Achilles heel of drug-target networks. Nat Biotechnol. 2008; 26(9): 983–984. PubMed Abstract \| Publisher Full Text\n\nMolecular Operating Environment (MOE), 2011.10, Chemical Computing Group Inc., 1010 Sherbooke St. West, Suite#910, Montreal, QC, Canada, H3A 2R7, 2011. Reference Source\n\nWeininger D: SMILES, a chemical language and information system. 1. Introduction to methodology and encoding rules. J Chem Inf Comput Sci. 1988; 28(1): 31–36. Publisher Full Text\n\nUniProt Consortium. The Universal Protein Resource (UniProt) in 2010. Nucleic Acids Res. 2010; 38(Database issue): D142–D148. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHu Y, Bajorath J: Drug activity data. Zenodo. 2014. Data Source" }	[ { "id": "6235", "date": "15 Oct 2014", "name": "Steffen Renner", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors provide and interesting study on drug promiscuity. Increasing our understanding of this important topic is of high value for the advancement of drug discovery. The current study investigates drug promiscuity over time, i.e. the number of targets reported for drugs over time. The authors conclude that this number is surprisingly low, if only high quality interactions are considered. The manuscript is well written, scientifically sound, and the analyzed dataset is made publicly available. Some points for minor modifications: When I read the title my first impression was that the manuscript compares the promiscuity of drugs with respect to their release data, e.g. \"do more recent drugs show more or less promiscuity than older drugs\". I suggest to change the title to better reflect that the release date of the target information is analyzed rather than the release date of the drugs. Page 4, second column: The only definition of the promiscuity rate I found in the manuscript is the following: \"The total number of target annotations of a drug represented its promiscuity rate\". The Wikipedia definition of a rate is \"Rate (mathematics), a specific kind of ratio, in which two measurements are related to each other\", therefore it appears to me that another name might better reflect the meaning of the promiscuity rate, e.g. just \"promiscuity\". On the other hand it is not the promiscuity which is changing: it is the number of identified interactions. Therefore another name might be even better. I did not find an activity cut-off (e.g. 10μM?) used for the activities in ChEMBL. Please add this information.", "responses": [ { "c_id": "1041", "date": "20 Oct 2014", "name": "Jürgen Bajorath", "role": "Author Response F1000Research Advisory Board Member", "response": "We thank the reviewer for his comments and the points raised.\"Title\": We would prefer retaining the current concise title. In our view, it reflects the time course of the promiscuity analysis. \"Rate\": From a mathematical/statistical point of view, the reviewer is correct (\"rate\" reflects a ratio). However, outside statistics, the term \"rate\" is often used synonymously with \"degree\", which is the intended meaning here. In previous publications, we have used both terms in the context of promiscuity analysis. We agree that the use of the term \"degree\" would avoid a potential inconsistency and should be preferred. \"Activity cut-off\": A cut-off value has not been applied in this analysis because the promiscuity time course was compared on the basis of low- vs. high-confidence activity data. The preferred use of high-confidence data would make the application of activity cut-off values a matter of debate. In a previous study (reference 11), it was shown that representative promiscuous compounds identified on the basis of high-confidence activity data are rarely weakly potent against their targets (which we consider an interesting observation).Many thanks again to both referees for taking the time to review this manuscript" } ] }, { "id": "6398", "date": "16 Oct 2014", "name": "Stefan A Laufer", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nKey points:500+ drugs successfully mapped to ChEMBL to follow a time course of promiscuity. When high-confidence activity data were considered, only small average increase in drug promiscuity over time were observed (from ca 1.5-3). When the stringency of data selection criteria was gradually relaxed and low-confidence data were considered, unrealistic increases in promiscuity over time were detected. As an illustration, please, see the imatinib example in Figure 9 (in comparison to Figure 8a) that the reader might find interesting.In addition, please, see the conclusions, especially the second part beginning with \" On the basis of high- confidence activity data ...\"", "responses": [] }, { "id": "6405", "date": "24 Oct 2014", "name": "Michael Walters", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have presented an interesting and careful study on drug promiscuity as analyzed through available activity databases. The authors also illustrate the different conclusions that can be drawn based on the quality of the data analyzed. The discrepancies they point out highlight the importance of high-quality data when evaluating the promiscuity of drugs. The manuscript is clear and well-written, the methods are scientifically sound, and the analysis is thorough and clearly presented. Comments: “Compounds with multiple Ki or IC50 measurements for the same target were retained if all these values fell within the same order.\" Should this read “…the same order of magnitude.”? “Surprisingly, for 282 drugs (~54%), no increase in promiscuity was detected on the basis of high-confidence activity records.” It would increase the impact of this article if the authors explained why this finding is surprising. For example, do these compounds represent drugs that were highly optimized for a single target? “There were only 14 drugs with an increase in promiscuity rates by 10 or more targets.“ As above, the authors might want to add their thoughts as to why these drugs have such high promiscuity rates. For example, are they kinase inhibitors which had known broad kinase panel activity and the complete scope of their kinase activity is only now coming to light? Suggestions for minor modifications: The dataset has been made publicly available through a DOI link. The link was broken when I attempted to follow it and look at the data. This link should be fixed.The authors have not provided any summary of the criteria used to determine what constitutes activity of a compound at a target (e.g. 10 μM cut-off? 100 μM?). If a strict cut-off cannot be given for each dataset, it is suggested that authors reported the range of activities reported in each dataset (one for the high-confidence data set, and one for each of the two low-confidence data sets). The authors mention that all the compounds were converted into SMILES strings during data analysis. It is suggested that the authors screen the SMILES list against a REOS/PAINS/etc. filters. It would be interesting to see if any of the high promiscuity compounds could be flagged as having substructures that could be responsible for non-specific activity. Or would have impeded the development of these drugs had they been prepared after cheminformatic filtering came into vogue. These suggested modifications would help increase the impact of this manuscript and could be of great interest to the drug discovery community.", "responses": [] } ]	1	https://f1000research.com/articles/3-218
https://f1000research.com/articles/3-264/v1	03 Nov 14	{ "type": "Research Article", "title": "Characterization of vaccine antigens of meningococcal serogroup W isolates from Ghana and Burkina Faso from 2003 to 2009", "authors": [ "Emma Ispasanie", "Gerd Pluschke", "Abraham Hodgson", "Ali Sie", "Calman MacLennan", "Oliver Koeberling", "Emma Ispasanie", "Gerd Pluschke", "Abraham Hodgson", "Ali Sie", "Oliver Koeberling" ], "abstract": "Neisseria meningitidis is a major cause of bacterial meningitis and a considerable health problem in the 25 countries of the ‘African Meningitis Belt’ that extends from Senegal in West Africa to Ethiopia in the East. Approximately 80% of cases of meningococcal meningitis in Africa have been caused by strains belonging to capsular serogroup A. After the introduction of a serogroup A conjugate polysaccharide vaccine, MenAfriVac™, that began in December 2010, the incidence of meningitis due to serogroup A has markedly declined in this region. Currently, serogroup W of N. meningitidis accounts for the majority of cases. Vaccines based on sub-capsular antigens, such as Generalized Modules for Membrane Antigens (GMMA), are under investigation for use in Africa. To analyse the antigenic properties of a serogroup W wave of colonisation and disease, we investigated the molecular diversity of the protein vaccine antigens PorA, Neisserial Adhesin A (NadA), Neisserial heparin-binding antigen (NHBA) and factor H binding protein (fHbp) of 31 invasive and carriage serogroup W isolates collected as part of a longitudinal study from Ghana and Burkina Faso between 2003 and 2009. We found that the isolates all expressed fHbp variant 2 ID 22 or 23, differing from each other by only one amino acid, and a single PorA subtype of P1.5,2. Of the isolates, 49% had a functional nhbA gene and 100% had the nadA allele 3, which contained the insertion sequence IS1301 in five isolates. Of the W isolates tested, 41% had high fHbp expression when compared with a reference serogroup B strain, known to be a high expresser of fHbp variant 2. Our results indicate that in this collection of serogroup W isolates, there is limited antigenic diversification over time of vaccine candidate outer membrane proteins (OMP), thus making them promising candidates for inclusion in a protein-based vaccine against meningococcal meningitis for Africa.", "keywords": [ "Neisseria meningitidis", "meningococcus", "meningitis", "serogroup W", "factor H binding protein", "NadA", "NHBA" ], "content": "Introduction\n\nNeisseria meningitidis is a major cause of bacterial meningitis in the African Meningitis Belt1. Between 1993 and 2012, nearly 1 million suspected cases were reported with 100,000 deaths, and 80% of the cases were caused by serogroup A2. Following the introduction of the serogroup A polysaccharide conjugate vaccine MenAfriVac™ in 2010, the incidence of group A disease decreased, but outbreaks of meningitis due to other meningococcal serogroups, in particular serogroup W, continue to occur1,3. Serogroup W was responsible for an epidemic of around 13,000 cases of meningitis in Burkina Faso in 20024 and contributed to a total of 639 deaths in 2012 in the same country5. Around 40% of infected people who develop sepsis die and survivors often suffer from limb loss, cognitive dysfunction, brain damage or visual impairment.\n\nAn approach towards developing a broadly-protective meningococcal vaccine for Africa is based on the use of subcapsular antigens included in GMMA (Generalized Modules for Membrane Antigens). GMMA are outer membrane blebs from bacteria genetically engineered to release large quantities of membrane vesicles, which are enriched in outer membrane proteins. Other strain modifications are included to increase safety and immunogenicity by the up-regulation of immunogenic antigens6,7. GMMA from genetically-engineered strains with up-regulated expression of meningococcal factor H binding protein (fHbp) have been shown to provide broad protection against African meningococcal isolates from different serogroups7. Other outer membrane antigens that have been shown to induce the production of bactericidal antibodies include PorA, Neisserial adhesin A (NadA)8 and Neisserial heparin-binding antigen (NHBA)9.\n\nTo help determine the potential coverage of these antigens in a GMMA-based vaccine for Africa, we investigated their genetic diversity in serogroup W carriage and disease isolates from Burkina Faso and Ghana collected between 2003 and 2009. These two countries have suffered repeatedly from meningococcal meningitis outbreaks4,10. Focusing on isolates collected over a period of years from a defined geographic region provides the opportunity to monitor the dynamics, variation and diversity of surface-exposed antigens over time.\n\n\nMaterials and methods\n\nThe N. meningitidis isolates investigated in this study were collected in the Kassena-Nankana District (KND) of Ghana and in the Nouna Health District (NHD) in the Kossi region of Burkina Faso. Case strains were isolated from the cerebrospinal fluid of meningitis patients, and carriage strains were isolated from throat swabs collected in the context of longitudinal carriage surveys. Isolation and characterization of the strains has previously been described10–13. Ethical clearance was obtained from the Ethics Committee of the War Memorial Hospital/Navrongo Health Research Centre in Ghana and the Ministry of Health and Local Ethics Committee of the Centre de Recherche en Santé de Nouna in Burkina Faso. Informed consent was obtained from all study participants. The 31 N. meningitidis carriage (n=21) and disease isolates (n=10) used in this study are described in the Table 1. The isolates were collected from Burkina Faso (n=8) and Ghana (n=23) during the period 2003–2009. The isolates were stored frozen in 10% skimmed milk at -80°C until analysis. The isolates were molecularly characterized with respect to fHbp, porA variable regions (VR), nadA and nhbA genes by sequencing. A subset of these isolates was also analysed for their fHbp expression level.\n\nMolecular characterization was performed on these isolates by PCR amplification and sequencing of fHbp, porA, nadA and nhbA.\n\n*Isolate used for fHbp protein expression analysis.\n\nNA: Inactive nadA due to insertion sequence IS1301.\n\nS: nhba gene with stop codon.\n\nY: Full length nhba gene\n\nN: No gene product obtained by PCR\n\nThe selected strains were sub-cultured on GC agar plates (Becton Dickinson, Franklin Lakes, NJ, USA) and incubated overnight at 37°C, 5% CO2. A loop-full of cells was resuspended in 500 μL sterile water and boiled for 10 minutes. The samples were pelleted at 17,900 g for 5 minutes in a microcentrifuge (Eppendorf). Genomic DNA was purified using an Invitrogen PureLink Genomic DNA kit (Invitrogen, San Diego, California, USA) according to the manufacturer’s instructions. The genes encoding fHbp, PorA VRs, NadA and NHBA were PCR amplified from all isolates using the primers described in Table 2. The final PCR reaction contained: 0.5 mM deoxynucleotide triphosphates, 5 U/mL Taq DNA polymerase, 1× Thermopol Reaction buffer (all New England BioLabs, Ipswich, USA), 1 µM primer solution (Sigma-Aldrich, St. Louis, Missouri, USA) and 100 ng of genomic DNA quantified with a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, USA). The PCR was performed using the Applied Biosystems GeneAmp PCR System 9700 (Applied Biosystems, Foster City, USA) with maximum ramping speeds using conditions described in Table 3. PCR products were separated by gel electrophoresis using a 0.8% Tris base, acetic acid and EDTA (TAE) agarose gel (BioRad Laboratories, Hercules, USA). PCR products were purified using the PureLink PCR Purification Kit (Invitrogen) according to the manufacturer’s instructions. The DNA amount was measured using the NanoDrop ND-1000 Spectrophotometer.\n\nFw: forward; Rv: reverse.\n\nThe primers used for porA VR1 sequencing were 210 and 103L (Table 2). We designed primers EI and H for sequencing of the VR2 region, by aligning the conserved regions upstream and downstream of VR2 using the alignment program Clustal W (http://www.ebi.ac.uk/Tools/msa/clustalw2/). PorA sequences from the following strains of different serogroups were used for the alignment: MC58 (GenBank accession number AE002098.2), Z2491 (AL157959.1), 053442 (CP000381.1), FAM18 (AM421808.1), M6190 (AEQF01000026.1), M13399 (AEQG01000023.1) and alpha 14 (AM889136.1) using Uniprot. The sequences were read at the Novartis Vaccines-Cellular Microbiology and Bioinformatics Unit Automated DNA Sequencing Facility, Siena, Italy, on an ABI 3730 DNA Analyzer. Sequences were analyzed using the Simmonics program (version 1.6) and Chromas (version 2.01). fHbp ID, porA VR and nhbA alleles were identified using the online Neisseria Sequence Typing database (http://pubmlst.org/neisseria). nadA sample and reference sequences were exported into the MEGA software package (version 5)14 and aligned for the construction of phylogenetic trees using the maximum likelihood method with the general time-reversible model of evolution and correction for partial deletion of gaps (GenBank accession numbers: nadA1 FJ619641.1; nadA2 GQ302859.1; nadA3 JN166979.1; nadA4 FJ619644.1; nadA5 FJ619645.1). All trees were un-rooted.\n\nFor 17 isolates labelled with an asterisk in Table 1, Western blot analysis of the fHbp expression level in whole cell samples was performed as described by Seib et al.15. The strains were sub-cultured on GC agar and incubated overnight at 37°C with 5% CO2. A 7 mL aliquot of Mueller-Hinton broth (Becton Dickinson, Franklin Lakes, NJ, USA) supplemented with 0.25% glucose (Sigma-Aldrich) was inoculated with single colonies to an optical density at 600 nm (OD600) of 0.12–0.16. The suspensions were incubated at 37°C with 5% CO2 to an OD600 of 0.6 corresponding to approximately 1.8×108 cfu/ml (exponential growth phase). The cells from 1 mL of culture were collected by centrifugation at 17,900 g for 5 min in a microcentrifuge (Eppendorf), re-suspended in 100 μL phosphate buffered saline (PBS) and heat inactivated in a water bath at 56°C for 1 hour. Protein concentrations of the lysates were determined using a Lowry protein assay kit (BioRad Laboratories, Hercules, USA) with bovine serum albumin (Sigma-Aldrich) as a standard. The fHbp amounts were estimated by SDS-PAGE and Western Blot. To 100 µL heat inactivated sample we added 100 µL SDS sample buffer (Invitrogen) and 10 µL of each sample was loaded on the gel. Recombinant fHbp (rfHbp) v.2 of 500, 250, 125 and 60 ng was used as standard. Positive and negative controls were whole cell lysates from N. meningitidis group B strain 8047 expressing fHbp v.2 and the isogenic fHbp knock-out mutant. Proteins were transferred to a nitrocellulose membrane (Invitrogen) using the iBlot system (Invitrogen). After blocking overnight in 3% milk powder in PBS (Merck, Whitehouse station, NJ, USA) at 4°C, fHbp proteins were detected with 1 μg/mL anti-fHbp mouse monoclonal antibody JAR31 (IgG2b) raised against recombinant fHbp v.3 ID 28, which shows cross-reactivity against most fHbp v.2 peptides16. The secondary antibody used was 1μg/mL of a horseradish peroxidase-labelled anti-mouse IgG (Invitrogen). The membranes were developed using SuperSignal WestPico Chemiluminescent Substrate (ThermoScientific, Waltham, Massachusetts, USA) according to manufacturer’s instructions, and the signal was detected with Amersham Hyperfilm ECL (GE Healthcare, Little Chalfont, UK). The amount of fHbp expressed by each isolate compared to the standard rfHbp was determined by densitometric analysis for three biological replicates using the ImageQuant 400 gel documentation system (GE Healthcare). The expression of fHbp by the test isolates was reported as percentages of the amount of fHbp expressed by bacterial cells compared to the reference strain, known to express relatively high amounts of fHbp v.217.\n\n\nResults\n\nPorA is an immunodominant antigen in N. meningitidis, but multiple subtypes exist with little cross-protection between meningococci expressing different PorA subtypes. fHbp can be divided into three antigenic variants, each of which is divided into sub-variants. Individual sequences are classified by a peptide ID number. Within each variant group, cross-protection is observed18. From the typing analysis of the fHbp and porA genes, all serogroup W isolates tested expressed fHbp variant 2, ID 22 (isolates from Burkina Faso) or 23 (isolates from Ghana), which differ by one amino acid, and PorA subtype P1.5,2 (Figure 1 and Supplemental File). Despite the limited number of isolates studied, these results suggest that between Burkina Faso and Ghana, which share a common border, there has been conservation of fHbp and PorA antigens among W isolates over a period of seven years.\n\nThe fHbp variant group is designated according to the classification proposed by Masignani et al.17. FHbp sequence ID, PorA subtype, nadA and nhbA allele were determined by sequence query on http://pubmlst.org/neisseria. Each isolate was typed by PCR amplification of each respective gene and sequence analysis using bioinformatics software Simmonics, Mega 5 and Chromas. NadA + IS1301: Strains with NadA encoding gene containing insertion sequence IS1301. NHBA-/stop codon: Strains lacking the NHBA encoding gene or having nhbA with stop codon.\n\nThe level of fHbp protein expression can affect susceptibility of meningococci to anti-fHbp antibodies. High expressers of fHbp are generally more susceptible to killing than low expressers19. We measured fHbp expression in 17 isolates. We selected 4 out of 8 (50%) strains from Burkina Faso and 13 out 23 (56%) strains from Ghana for fHbp expression analysis. These were selected to cover isolates from different years including the oldest and newest strains. Within this group of strains selected, n=2 (50%) of the strains from Burkina Faso and n=7 (53%) of the strains from Ghana were case isolates, while the remainder were carriage isolates. We prepared whole cell extracts of the serogroup W test strains and the serogroup B reference strain and compared fHbp levels with defined amounts of a fHbp v.2 protein standard by Western blot and densitometry measurement (Dataset 1). Expression level of the reference serogroup B strain 8047 was set to 100% and levels of expression of the serogroup W strains were compared with the reference strain. Isolates with means below 33% of the reference strain were classified as low expressers while isolates with expression above 100% were categorized as high expressers. Those with mean fHbp expression between 33–100% were considered intermediate expressers. The expression of fHbp among the W isolates was variable, ranging from 50–152%, compared to the reference serogroup B strain 8047, with 41% of the isolates expressing equal or higher levels of fHbp compared to the reference strain (Figure 2). There was no significant difference in fHbp expression between case and carrier isolates studied (P=0.74, Mann Whitney U test). This indicates that levels of fHbp protein on the bacterial surface can vary among strains collected from a relatively small region and expressing the same fHbp ID.\n\nBars represent the mean percentage from three biological replicates compared with the expression of fHbp of the reference group B strain 8047, a high expresser of fHbp variant 2 ID 77, which was set at 100%. Isolates with means below 33% were classified as low expressers while isolates with expression above 100% were categorized as high expressers. Values between 33–100% were considered as intermediate expression. Bars represent standard errors.\n\nNadA and NHBA induce the production of bactericidal antibodies against N. meningitidis serogroup B strains. Wang et al. found that nadA was not present among a small number (n=13) of W isolates tested as part of an analysis of 896 serogroup B, C, Y and W isolates from the USA, while nhbA was present in 92% of W isolates20. Among the African W strains investigated in this study, the nadA allele 3 was present in 26/31 (84%) of isolates (Figure 1). Among the remaining five W isolates (1 case, 4 carrier isolate), PCR amplification across the nadA site gave a 2 kb product instead of the expected 1 kb product. Western blotting using whole cell lysate and polyclonal mouse anti-NadA allele 3 antibody indicated that these isolates did not express NadA (Dataset 1). Sequencing of this fragment confirmed the presence of the insertion sequence IS1301. This 842-bp mobile genetic element is known to cause a number of effects including insertions and deletions that result in silent mutations, knock-out of gene expression or regulation of downstream-located genes. For example, insertion of IS1301 into the capsular siaA gene mediates loss of encapsulation resulting in increased adherence and entry of meningococci into epithelial cells21,22. 17 out of 21 carrier (81%) and 9 out of 10 (90%) case isolates had a nadA gene. Previous reports found that nadA is present in about 50% of group B case isolates, but underrepresented in carrier isolates23.\n\nNhbA was present in 30/31 (94%) of the meningococcal isolates studied. However, genetic sequencing in these isolates revealed a stop codon for 15/30 isolates, which has not previously been reported. The alleles of the remaining strains were identified as allele 17 (Supplemental File) using the Neisseria typing database available at http://pubmlst.org/neisseria/NHBA/.\n\n\nDiscussion\n\nSince the introduction of a meningococcal A polysaccharide conjugate vaccine MenAfriVac® in the African Meningitis Belt, outbreaks of meningitis caused by non-serogroup A meningococci, particularly W, are occurring with increased frequency. The development of a protein-based vaccine that can provide broad protection is an attractive prospect. An approach to understanding whether protein-based vaccines could have an impact on reducing the burden of meningococcal disease in the African Meningitis Belt, is to examine the genetic diversity of carriage and disease isolates of serogroup W. In this study, we focused on investigating the molecular diversity of four OMP vaccine antigens of 31 carriage and disease isolates of serogroup W from Ghana and Burkina Faso. The strains studied were isolated between 2003 and 2009 and contain conserved fHbp, porA and nadA genes, suggesting little antigenic diversification over time. A stop codon was identified among over half of the nhbA genes sequenced and was associated with a lack of expression of NHBA protein.\n\nPreviously, Pajon et al. performed a molecular characterization of 106 invasive meningococcal isolates from 13 African countries, 26 of which were from Burkina Faso and 3 from Ghana. Of the serogroups W analysed in the study, 58% were fHbp variant 2, in common with all W isolates from our collection, while 34% were variant 1 and 8% variant 3. Concordant with our findings, 98% of W were PorA subtype P1.5,2 or a related subtype indicating a marked homogeneity of PorA type among African serogroup W isolates19. A more recent longitudinal study found that a hypervirulent ST-11 serogroup W clone was responsible for most meningococcal disease in 2011 and 201224. All the isolates expressed PorA 1.5,2 and 96.4% had FetA (iron-regulated outer-membrane protein which is involved in uptake of siderophores25) variant F1-1. In accordance with our study, these two previous studies emphasise the limited diversification of major OMPs in the serogroup W meningococcal population in Africa. Studies with isogenic mutants with different expression levels of fHbp suggested that low fHbp expression contributes to resistance to anti-fHbp bactericidal activity19. It has been suggested that sparse distribution of antigens on the bacterial surface impedes cross-linking of two IgG anti-fHbp antibodies to correctly spaced epitopes26. Consequently, the antibodies cannot engage the complement protein C1q, preventing activation of the classical complement pathway. In the present study, the serogroup W isolates were found to express medium to high levels of fHbp when compared with a serogroup B strain known to express naturally relatively high levels of fHbp17, and there was no significant difference in expression between case and carrier isolates. This, together with the conservation of the fHbp ID among carrier and case isolates indicates that both carrier and case isolates could be targets of vaccine-induced anti-fHbp antibodies. NadA has emerged as an important protein for adhesion and invasion, and has been shown to elicit bactericidal antibodies8. In this study, the presence of the nadA gene in most case and carrier strains isolated from African countries suggests that NadA could be a potentially important vaccine antigen to be included in a GMMA vaccine for Africa.\n\n\nConclusion\n\nThis longitudinal study of meningococcal serogroup W isolates from two African countries, together with the findings of other studies, suggests that there is limited antigenic variation of meningococcal outer membrane proteins that induce bactericidal antibodies. These findings support a strategy of using protein-based vaccines, such as GMMA, to prevent meningococcal meningitis in Africa caused by serogroup W.\n\n\nData availability\n\nF1000Research: Dataset 1. Data of fHbp and NadA expression in serogroup W isolates from Ghana and Burkina Faso, 10.5256/f1000research.3881.d3632628", "appendix": "Author contributions\n\n\n\nOK and CAM conceived the study. OK designed the experiments. EI carried out the research. EI, OK and CAM wrote the manuscript. AH, AS and GP provided the group W isolates studied. All authors were involved in the reviewing of the manuscript and have agreed to its final content.\n\n\nCompeting interests\n\n\n\nOK and CAM are both employees of the Novartis Vaccines Institute for Global Health. CAM has received grant support from GlaxoSmithKline.\n\n\nGrant information\n\nThis work was supported by an EU FP7 Marie Curie Actions Industry Academia Partnerships and Pathways (IAPP) Consortium Programme, entitled GENDRIVAX (Genome-driven vaccine development for bacterial infections).\n\n\nAcknowledgments\n\nWe would like to thank Dan Granoff for providing the monoclonal antibody JAR31 used in this study.\n\n\nSupplementary material\n\nSupplemental file. Sequencing analysis of fHbp, porA, nhbA and nadA genes from serogroup W strains.\n\nThe genes encode fHbp v.2, ID 22 or 23, PorA subtype P1.5,2, NHBA allele 17 and NadA allele 3 with and without insertion sequence IS1301. DNA sequences were translated into protein sequences and sequence queries were performed using the database on http://pubmlst.org/neisseria.\n\nClick here to access the data.\n\nhttp://dx.doi.org/10.5256/f1000research.3881.s37766\n\n\nReferences\n\nTeyssou R, Muros-Le Rouzic E: Meningitis epidemics in Africa: a brief overview. Vaccine. 2007; 25(Suppl 1): A3–A7. PubMed Abstract \| Publisher Full Text\n\nCampagne G, Schuchat A, Djibo S, et al.: Epidemiology of bacterial meningitis in Niamey, Niger, 1981–96. Bull World Health Organ. 1999; 77(6): 499–508. PubMed Abstract \| Free Full Text\n\nCollard JM, Maman Z, Yacouba H, et al.: Increase in Neisseria meningitidis serogroup W135, Niger, 2010. Emerg Infect Dis. 2010; 16(9): 1496–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoumare B, Ouedraogo-Traore R, Sanou I, et al.: The first large epidemic of meningococcal disease caused by serogroup W135, Burkina Faso, 2002. Vaccine. 2007; 25(Suppl 1): A37–A41. PubMed Abstract \| Publisher Full Text\n\nWHO, Weekly Epidemiology Record. 2013; 88. : 129–36.\n\nBerlanda SF, Colucci AM, Maggiore L, et al.: High yield production process for Shigella outer membrane particles. PLoS One. 2012; 7(6): e35616. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoeberling O, Ispasanie E, Hauser J, et al.: A broadly-protective vaccine against meningococcal disease in sub-Saharan Africa based on generalized modules for membrane antigens (GMMA). Vaccine. 2014; 32(23): 2688–95. PubMed Abstract \| Publisher Full Text\n\nComanducci M, Bambini S, Brunelli B, et al.: NadA, a novel vaccine candidate of Neisseria meningitidis. J Exp Med. 2002; 195(11): 1445–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSerruto D, Spadafina T, Ciucchi L, et al.: Neisseria meningitidis GNA2132, a heparin-binding protein that induces protective immunity in humans. Proc Natl Acad Sci U S A. 2010; 107(8): 3770–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeimkugel J, Hodgson A, Forgor AA, et al.: Clonal waves of Neisseria colonisation and disease in the African meningitis belt: eight- year longitudinal study in northern Ghana. PLoS Med. 2007; 4(3): e101. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGagneux S, Hodgson A, Ehrhard I, et al.: Microheterogeneity of serogroup A (subgroup III) Neisseria meningitidis during an outbreak in northern Ghana. Trop Med Int Health. 2000; 5(4): 280–7. PubMed Abstract \| Publisher Full Text\n\nLeimkugel J, Forgor AA, Dangy JP, et al.: Genetic diversification of Neisseria meningitidis during waves of colonization and disease in the meningitis belt of sub-Saharan Africa. Vaccine. 2007; 25(Suppl 1): A18–A23. PubMed Abstract \| Publisher Full Text\n\nSie A, Pfluger V, Coulibaly B, et al.: ST2859 serogroup A meningococcal meningitis outbreak in Nouna Health District, Burkina Faso: a prospective study. Trop Med Int Health. 2008; 13(6): 861–8. PubMed Abstract \| Publisher Full Text\n\nTamura K, Peterson D, Peterson N, et al.: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011; 28(10): 2731–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSeib KL, Serruto D, Oriente F, et al.: Factor H-binding protein is important for meningococcal survival in human whole blood and serum and in the presence of the antimicrobial peptide LL-37. Infect Immun. 2009; 77(1): 292–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBeernink PT, Welsch JA, Bar-Lev M, et al.: Fine antigenic specificity and cooperative bactericidal activity of monoclonal antibodies directed at the meningococcal vaccine candidate factor H-binding protein. Infect Immun. 2008; 76(9): 4232–40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMasignani V, Comanducci M, Giuliani MM, et al.: Vaccination against Neisseria meningitidis using three variants of the lipoprotein GNA1870. J Exp Med. 2003; 197(6): 789–99. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSeib KL, Brunelli B, Brogioni B, et al.: Characterization of diverse subvariants of the meningococcal factor H (fH) binding protein for their ability to bind fH to mediate serum resistance, and to induce bactericidal antibodies. Infect Immun. 2011; 79(2): 970–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPajon R, Fergus AM, Koeberling O, et al.: Meningococcal factor H binding proteins in epidemic strains from Africa: implications for vaccine development. PLoS Negl Trop Dis. 2011; 5(9): e1302. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang X, Cohn A, Comanducci M, et al.: Prevalence and genetic diversity of candidate vaccine antigens among invasive Neisseria meningitidis isolates in the United States. Vaccine. 2011; 29(29–30): 4739–44. PubMed Abstract \| Publisher Full Text\n\nHilse R, Hammerschmidt S, Bautsch W, et al.: Site-specific insertion of IS1301 and distribution in Neisseria meningitidis strains. J Bacteriol. 1996; 178(9): 2527–32. PubMed Abstract \| Free Full Text\n\nHilse R, Stoevesandt J, Caugant DA, et al.: Distribution of the meningococcal insertion sequence IS1301 in clonal lineages of Neisseria meningitidis. Epidemiol Infect. 2000; 124(2): 337–40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCapecchi B, Adu-Bobie J, Di Marcello F, et al.: Neisseria meningitidis NadA is a new invasin which promotes bacterial adhesion to and penetration into human epithelial cells. Mol Microbiol. 2005; 55(3): 687–98. PubMed Abstract \| Publisher Full Text\n\nKristiansen PA, Ba AK, Sanou I, et al.: Phenotypic and genotypic characterization of meningococcal carriage and disease isolates in Burkina Faso after mass vaccination with a serogroup a conjugate vaccine. BMC Infect Dis. 2013; 13: 363. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBeucher M, Sparling PF: Cloning, sequencing, and characterization of the gene encoding FrpB, a major iron-regulated, outer membrane protein of Neisseria gonorrhoeae. J Bacteriol. 1995; 177(8): 2041–9. PubMed Abstract \| Free Full Text\n\nWelsch JA, Ram S, Koeberling O, et al.: Complement-dependent synergistic bactericidal activity of antibodies against factor H-binding protein, a sparsely distributed meningococcal vaccine antigen. J Infect Dis. 2008; 197(7): 1053–61. PubMed Abstract \| Publisher Full Text\n\nFeavers IM, Maiden MC: A gonococcal porA pseudogene: implications for understanding the evolution and pathogenicity of Neisseria gonorrhoeae. Mol Microbiol. 1998; 30(3): 647–56. PubMed Abstract \| Publisher Full Text\n\nIspasanie EHB, Pluschke G, et al.: Dataset 1: Data of fHbp and NadA expression in serogroup W isolates from Ghana and Burkina Faso. F1000Research. 2014. Data Source" }	[ { "id": "7910", "date": "10 Mar 2015", "name": "Georgina Tzanakaki", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, Ispasanie et al. describes the characterization of vaccine antigens of meningococcal serogroup W isolates from both carriers and patients in Ghana and Burkina Faso.The work seems very interesting and adds up to the previous knowledge with information on the limited antigenic variation of meningococcal OMPs that induce antibodies. The manuscript is well written and the methodology is well defined.Minor comments:The authors state in the discussion section, that after the introduction of the meningococcal A vaccine outbreaks caused particularly by serogroup W are occurring with increased frequency. To the reader’s surprise, only 31 isolates were studied (21 carrier and 10 patient strains) within a seven year period (2003-2009). Maybe the authors would consider adding more information of the numbers of serogroup W incidence or number of cases in both countries and the reason why they choose only this limited number for characterization of the vaccine antigens.", "responses": [] }, { "id": "8120", "date": "07 Apr 2015", "name": "Kate L. Seib", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article describes the molecular characterization of the diversity of PorA, fHbp, NadA and NHBA vaccine antigens in 31 serogroup W Neisseria meningitidis strains (21 carriage and 10 disease strains, isolated from Ghana and Burkina Faso between 2003 and 2009). The article is clear and well written and provides information to help guide vaccine development for serogroup W meningococcal disease in these countries. Some comments and questions include:Only 10 serogroup W disease isolates were investigated. How many serogroup W cases were there in this region between 2003-2009, and how many have there been post MenAfriVac implementation in 2010? Is there any reason to expect a change in serogroup W epidemiology post MenAfriVac implementation, given that the total number of serogroup W cases has increased, as well as the variability of meningococcal epidemiology?It is stated that “a stop codon was identified among over half of the nhbA genes sequenced and was associated with a lack of expression of NHBA protein”. Where is the premature stop codon located in the subset of nhba genes? Was expression examined by Western blot? I could not access sequence data from the file “Supplemental file. Sequencing analysis of fHbp, porA, nhbA and nadA genes from serogroup W strains.”", "responses": [] } ]	1	https://f1000research.com/articles/3-264
https://f1000research.com/articles/3-224/v2	31 Oct 14	{ "type": "Research Note", "title": "Flu vaccine experiences and beliefs influence vaccination decision making more than knowledge", "authors": [ "Dara Whalen", "David Molnar", "Faye Milne", "Lauren Schwal", "Virginia Hackett", "Jonathan Coffman", "Dara Whalen", "David Molnar", "Faye Milne", "Lauren Schwal", "Virginia Hackett" ], "abstract": "Mass immunization programs have proven to be a primary preventive measure to limit the spread of many infectious diseases worldwide. Nurses are trained to be leaders in preventing potential global health problems, but they are one of the groups with the lowest rates of compliancy in receiving influenza vaccination. Since nursing faculty are important role models in molding attitudes and behaviors of future nurses (their students), we set out to explore the knowledge, attitudes and beliefs of the nursing faculty and their students regarding influenza vaccine decision making. Our study included an assessment of the knowledge, attitudes and beliefs (KABs) of the nursing faculty and students related to influenza vaccination and whether their KABs influence the decision to receive or decline the vaccination. A cross-sectional survey was conducted using an anonymous questionnaire and our study indicated that personal experiences—either positive or negative—had a direct effect on influenza vaccine decision-making. Additionally, personal experiences influenced beliefs, and beliefs were shown to influence decision-making regarding vaccination. While beliefs and personal experiences had a direct effect on vaccine decision-making, knowledge had only an indirect effect through beliefs. Our study demonstrated that even though nursing practice is supposed to be driven by evidence-based medical practices, personal practices by nurses may be more influenced by personal beliefs than medical knowledge.", "keywords": [ "The influenza virus is a well-known infectious pathogen that causes annual epidemics and pandemics occurring at 10 to 50 year intervals1. The spread of influenza could be ameliorated or diminished with an appropriate level of herd immunity2", "3", "however", "individual attitudes and beliefs are known to influence a healthcare worker’s decision to receive an influenza vaccination4. Although nurses are trained to be leaders in preventing potential global health problems", "research has shown that they are one of the patient-exposed groups with the lowest rates of compliance in receiving influenza vaccination5. Nursing faculty are important role models in molding attitudes and behaviors of future nurses (their students) but there is a paucity of research exploring the knowledge", "attitudes", "and beliefs of nursing faculty members and their students regarding influenza immunization and compliance practices." ], "content": "Introduction\n\nThe influenza virus is a well-known infectious pathogen that causes annual epidemics and pandemics occurring at 10 to 50 year intervals1. The spread of influenza could be ameliorated or diminished with an appropriate level of herd immunity2,3, however, individual attitudes and beliefs are known to influence a healthcare worker’s decision to receive an influenza vaccination4. Although nurses are trained to be leaders in preventing potential global health problems, research has shown that they are one of the patient-exposed groups with the lowest rates of compliance in receiving influenza vaccination5. Nursing faculty are important role models in molding attitudes and behaviors of future nurses (their students) but there is a paucity of research exploring the knowledge, attitudes, and beliefs of nursing faculty members and their students regarding influenza immunization and compliance practices.\n\nAn appropriate theoretical framework to examine nurses’ decisions to receive the influenza vaccination was the Health Belief Model6. The framework was used due to its ability to predict health behaviors based on the theory that an individual’s willingness to change behaviors and follow health recommendations is determined by personal beliefs or perceptions about a disease7. In the context of this study, the theoretical framework helped to explain the relationship of nursing faculty and students’ perceptions and health behavior practices relative to influenza vaccination. This model has been cited in numerous studies related to the adoption of health care practices.\n\nBond and Nolan used the Health Belief Model to make sense of perceptions about disease severity and susceptibility risks related to the decision of parents to vaccinate their children8. The researchers indicated that the degree of familiarity with a disease (or lack of) and the characteristics related to the disease prompted preventive action. Similarly Toronto and Mullaney found that Registered Nurses (RNs) acknowledged fear of adverse reactions and uncertainty of efficacy of the current vaccine as primary barriers for being vaccinated9.\n\nA cross sectional survey explored the rate of influenza immunizations among health-care workers in a Saudi hospital and identified reasons for electing or declining the flu immunization10, and the most common reason for not being vaccinated was a belief that the vaccine was not effective in preventing the disease. Hunt reported that nursing students participated in H1N1 and seasonal vaccination based on their risk perception and history of previous illness as motivators for immunization while those who abstained perceived the vaccine as not being necessary11. Another cross sectional study of nursing students reported that only 43% had been vaccinated12 because the student nurses did not believe the seriousness and facts regarding the H1N1 influenza outbreak.\n\nOther researchers reported that nurses who had a better understanding of the risk of flu infection, who had knowledge of the disease, and who had been previously vaccinated were found to be more inclined to be vaccinated yearly13.\n\nA cross-sectional mail survey revealed that the most common reason for being vaccinated was the belief of self-protection and the most common reason for not being vaccinated was concern about adverse reactions14. Similarly, in the United Kingdom, general practitioners and practice nurses found that confidence in the efficacy of the vaccine along with the conviction of the severity of the influenza were predictors to the acceptance of the vaccine15.\n\nIn an urban community teaching medical center, health care workers were surveyed on their participation in influenza vaccination16. The main study findings were that (a) a higher knowledge score of influenza, and (b) the belief that the vaccine was to protect patients, were both correlated with increased health-care worker vaccination rates Group identification, professional responsibility and patient protection were also identified in another study17. The researchers identified in their study of 531 nurses in Switzerland that rejection of the flu vaccine occurred secondary to knowledge of the flu and the vaccine. Yet, in another study, only 64% of the 513 nurses surveyed intended to receive the influenza vaccination despite being exposed to educational bulletins and receiving information about influenza severity18.\n\nBy using the Health Belief Model to assess the knowledge, attitudes and beliefs (KABs) of the nursing faculty, students and public relative to influenza vaccination, we have concluded that experiences and beliefs influence vaccination decision making more than knowledge.\n\n\nMethods\n\nA cross-sectional survey was conducted using an anonymous questionnaire, and population samples of nursing faculty, nursing students and health-fair attendees were surveyed with questions related to their attitudes and beliefs surrounding yearly vaccination against seasonal influenza, and answers were documented in a paper format. The participants for this research project were recruited from three sources.\n\nThe first source was nursing faculty and students attending an annual research conference in March, 2011. The sponsoring organization, agreed to allow the recruitment of participants at this conference. A convenience sampling method was used to ask nursing faculty members, students, and nursing community members present at the conference to complete a survey related to their attitudes towards and beliefs surrounding yearly vaccination against seasonal influenza. An announcement was made at the opening of the conference explaining the purposes of this study and asking for participation by completing an anonymous survey. The survey instrument used was developed by researchers at the University of Michigan and modified, with permission from the authors, to address the research questions and population of interest for this study. The 24-item survey included demographic information and 22 closed-ended and 2 open-ended questions. The survey instrument was designed to measure knowledge, attitudes and beliefs (KABs) regarding the seasonal influenza vaccine. Practices related to influenza vaccination, receipt and recommendations were also measured. A blank copy of the survey and an attached cover letter describing the study and requesting participation was distributed to all attendees during the opening session of the conference. The surveys were distributed during the opening session of the conference and took approximately 30 minutes to complete based on observation. A total of 1,000 surveys were distributed to nursing faculty and nursing students and 226 surveys were collected. The completed surveys were placed in sealed boxes by the responders.\n\nA second source of sampling came from nursing students and community members attending a health fair in South Florida. Nursing students and community members were surveyed and data were collected and compiled for statistical analysis.\n\nCommunity members not attending the health-fair were also surveyed and all documented data were collected and compiled for statistical analysis. Community members were chosen randomly in order to provide a comparison of medically educated professionals—nursing and faculty—to the general public.\n\nThe conceptual framework for the statistical analysis was classical test theory19. The disease detection model was used to evaluate the predictive success of the model, and logistic regression was the statistical method used to estimate the probability of receiving the vaccine, based on predictors (knowledge, beliefs and the influence of personal experiences). Logistic regression was better suited than linear regression because the dependent variable is dichotomous.\n\nA logistic regression model was evaluated in three steps. First, the extent to which the model fits the observed data was evaluated. Next, the statistical significance and effect size of individual predictors was evaluated. Since the scales for the knowledge index and the beliefs index have no natural interpretation like weight or temperature scales, the variables were standardized in order to make the scale of the variables more comparable. A standardized variable is transformed so its mean is zero and its standard deviation is one regardless of the units in which it was measured. In the third step, a classification table was generated that compares predicted outcomes to observed outcomes. Sensitivity and selectivity were calculated from the classification table.\n\n\nResults\n\nThe descriptive statistics for the study are summarized in Table 1. 29 nursing faculty, 197 nursing students and 152 community members residing in Broward and Miami-Dade counties in South Florida represented the study with ages ranging from 19 to over 65 with White non-Hispanics representing only 25% of the surveyed population. 37% described their ethnicity as Hispanic and 17% described their ethnicity as Black. The largest age group at 42% was 19–29 year olds and 60% of the total population was 40 years and younger.\n\n169 individuals received the vaccine and 209 did not. Based on a crosstabulation analysis, the proportion of nursing faculty (62%) receiving the vaccine was not significantly greater than the proportion of community members (49%) receiving the vaccine, Χ2=1.746, p=0.19; however, the faculty proportion was significantly greater than the proportion of nursing students (39%) receiving the vaccine, Χ2=5.480, p=0.02. Note that the achieved power of the analysis was only .63 due to the small number of nursing faculty in the sample.\n\nAccording the health benefits model, a person’s decision to participate in influenza vaccination is influenced by knowledge, beliefs and personal experiences6. As measured by Cronbach’s alpha, the reliability of the beliefs index was good, α=0.82, but the reliability of the knowledge index was poor, α=0.52. Furthermore, the knowledge index was highly correlated with the beliefs index, r=0.54. The poor reliability of the knowledge index reduced its power in the analysis. The collinearity with beliefs and the low power made it less likely that the knowledge index would be a statistically significant predictor of receiving the flu vaccine.\n\nLogistic regression was conducted to determine the extent to which the independent variables (influential personal experiences, vaccine beliefs index, and vaccine knowledge index) were predictors of receiving the flu vaccine. Personal positive or negative experiences were dichotomous depending on whether the respondent reported being influenced by positive or negative experiences. The knowledge scale had six items and the belief scale had four items. The belief and knowledge indexes were based on the sum of items on a 5-point Likert agreement scale. Data screening identified no outliers. Model fit statistics revealed that the model fitted the data since the Hosmer-Lemeshow Goodness-of-Fit test was not significant (-2 Log Likelihood = 371.427 and Hosmer-Lemeshow Goodness-of-Fit = 9.646, p=0.29). The Omnibus Test of Model Coefficients showed that the generated model was significantly different from the constant–only model, Χ2(4) = 136, p<0.001. In other words, compared to the prediction that everyone does not receive the vaccine (the most frequent outcome), the predictors improved the prediction. Wald statistics indicated that the knowledge index was not a significant predictor of the decision to vaccinate, Wald=0.075, p=0.78. Consequently, the knowledge index was dropped as a predictor in a revised, more parsimonious model.\n\nFor the revised model, (excluding knowledge index), the proportion correctly predicted went from 55.3% with the constant only model to 77.0%. The specificity (probability of true negative) is 80%, 95% CI [74%, 85%] and the sensitivity (probability of true positive) is 73%, 95% CI [66%, 80%], see Figure 1. Note in Figure 1 the left bar represents all respondents who actually received the vaccine and the right bar represents all who did not. The shading of the bars indicates whether the respondent was predicted to receive the vaccine (dark shading) or was predicted not to receive the vaccine (light shading). Regression coefficients are presented in Table 2. Wald statistics indicated that influential personal experiences and the beliefs index significantly predict receiving the flu vaccination. The odds ratio for influential positive experiences, OR = 4.49, 95% CI [2.71, 7.45], indicated a moderate effect size while the odds ratio for influential negative experiences, OR = 0.381, 95% CI [0.163, 0.892], indicated a smaller but still moderate effect size. The odds ratio for the beliefs index standardized, OR = 2.86, 95% CI [2.08, 3.94], indicated a moderate effect size similar to the effect size for influential negative experiences.\n\nNote. -2 Log Likelihood = 377.960, Χ2 (3) = 141.818, p<0.001. (N = 378).\n\nRespondents were nearly three times more likely to report they were influenced by positive experiences (n=153) as opposed to negative experiences (n=52).\n\nA 2 × 2 factorial ANOVA was conducted to evaluate the relationship between influential personal experiences with the flu vaccine and beliefs about receiving the flu vaccine. The independent variables, positive experiences and negative experiences, were dichotomous. The dependent variable was the standardized beliefs index. A preliminary analysis evaluating the homogeneity of variances assumption, using Levene’s test, indicated no significant difference in variances among groups, F(3,374) = 1.74, p = 0.16. The ANCOVA indicated no significant interaction between positive experiences and negative experiences, F (1,374) = 0.060, p = 0.81. There was a significant effect for influential positive experiences, F (1,375) = 51.2, p<0.001, and a significant effect for influential negative experiences, F (1,375) = 8.51, p = 0.004, see Table 3. Having an influential positive experience improves the belief index by two thirds of a standard deviation, d = 0.68, 95% CI (0.49, 0.86), indicating a large effect size. Having an influential negative experience reduces the belief index by approximately one third of a standard deviation, d = 0.40, 95% CI [0.13, 0.66], indicating a moderate effect size.\n\np<0.01 *p<0.001\n\nNote. df=degrees of freedom, SS=sum of squares, MS=mean square, F=value of F-statistic, and η2=eta squared\n\nThe results of this study were combined to establish a working model (Figure 2) that illustrated the variables and their influence on vaccine decision making. Our study indicated that personal experiences—either positive or negative—had a direct effect on whether or not a person received the influenza vaccination. Additionally, personal experiences influenced beliefs, and beliefs were shown to influence decision making regarding vaccination. Knowledge did not have a direct effect on the decision making process. However, knowledge is correlated with beliefs, r=.58, p<.001. The research design does not identify whether beliefs influence knowledge or knowledge influences beliefs.\n\n\nDiscussion\n\nAccording to our study and model, knowledge about the influenza vaccine was not a reliable predictor as to whether or not a person would actually participate in vaccination. In fact, it is likely that knowledge about influenza vaccination is mentally processed through a person’s belief system as a heuristic filter as indicated by other reports20. It is well-known that heuristics (mental short cuts or rules of thumb) can lead to erroneous decision-making even to the point where a person may later be embarrassed by his or her decision21. Many of the decisions to vaccinate, therefore, are not based on evidence-based science.\n\nIn a 2007 study, “Gaps between knowing and doing” a systematic review of English language studies involving human subjects, the researchers found that there was a significant gap between routine clinical practices and scientifically established clinical practice guidelines22. That finding was consistent with our study where we identified that beliefs influence vaccine decision making (Figure 2).\n\nPersonal experiences, on the other hand, were significant in our study to the point where a positive or negative experience influenced their decision to receive the vaccine, and the personal experiences also affected their belief. In the sample 95 respondents reported being influenced by a positive experience and 13 reported being influenced by a negative experience. A positive experience increased the odds of receiving the flu vaccination by 4.5 times, whereas, a negative experience decreased the odds of receiving the flu vaccine by only 0.38 times. It is possible that the positive experience confirmed the person’s knowledge that receiving the flu shot was important and helped alleviate any hesitation of receiving the flu shot due a preconceived fear of a negative outcome. Accordingly, it would be pertinent for the promotion of vaccination to focus only on positive personal experiences and spend less time on alleviating the fear of negative outcomes, those that are real or that are perceived as real. A focus group study on Human papillomavirus (HPV) vaccination among girls in Australia identified that social capital, peer acceptance and trust in authority were not only related to the likelihood of receiving the vaccine but could be influenced through social marketing campaigns23. Additionally, it is well known in marketing that an emphasis on timing is relevant to positive outcomes24.\n\nThe limitations of our study include a small sample size for the faculty and one that is localized to a nursing school in South Florida. The population of South Florida is one of cultural diversity and is quite distinct from other regions of the United States. The participants in our study reflected that distinctness with 54% of the respondents describing their ethnicity as either Black or Hispanic with another 16% describing themselves as “other”. Only 25% indicated that their ethnic background was White. The ethnic composition of the study is significant when understanding that a person’s belief is influenced by “repeated articulations” of life and culture25. Investigating cultural contributions to our model is an area for future research.\n\nMost campaigns to increase immunization rates among nurses are knowledge-based, but the lack of focus on attitudes and beliefs could contribute to the failure of such campaigns. The nursing practice is increasingly being driven by medical evidence but personal practices by those in the profession may not follow the same path. Utilizing a multidirectional approach addressing other domains of learning to influence vaccine decision-making may be more effective than traditional knowledge-based programs. Perhaps, an application component–such as the measuring of antibody titers–could be implemented in vaccination training to complement the knowledge component. A rise in antibody titers (levels) demonstrates the effectiveness of a vaccine, and the measurement of a rise in levels of antibodies would provide positive feedback to the trainee.\n\nRelative to our study in South Florida, we would recommend a social marketing campaign targeting the public, a campaign organized by the nursing faculty and one that would involve nursing students who would encounter positive experiences and a lack of negative experiences. The campaign would also focus on community support and trust among respected leaders in the community—including parents, healthcare workers, clergy, etc—with a positive emphasis on a call to action in a timely manner. An emphasis on positive experiences could have a dual objective: an increase in vaccination rates and a change in beliefs.\n\n\nData availability\n\nfigshare: Data Predicting Flu Vaccine Based on Experiences and Beliefs, http://dx.doi.org/10.6084/m9.figshare.109870526", "appendix": "Author contributions\n\n\n\nDW, DM, FM, LS, VH and JC collaborated and conceptualized the study. DW, FM, LS and VH surveyed and collected data from the nursing school, and JC surveyed and collected data from the public. DM statistically analyzed the data and JC wrote the article.\n\n\nCompeting interests\n\n\n\nNo conflicting interests were disclosed.\n\n\nGrant information\n\nThis work was funded by the College of Health Sciences at Barry University.\n\n\nReferences\n\nPotter CW: A history of influenza. J Appl Microbiol. 2001; 91(4): 572–579. PubMed Abstract \| Publisher Full Text\n\nPlans-Rubio P: The vaccination coverage required to establish herd immunity against influenza viruses. Prev Med. 2012; 55(1): 72–77. PubMed Abstract \| Publisher Full Text\n\nAnderson RM: The concept of herd immunity and the design of community-based immunization programmes. Vaccine. 1992; 10(13): 928–935. PubMed Abstract \| Publisher Full Text\n\nHofmann F, Ferracin C, Marsh G, et al.: Influenza vaccination of healthcare workers: a literature review of attitudes and beliefs. Infection. 2006; 34(3): 142–147. PubMed Abstract \| Publisher Full Text\n\nWalker FJ, Singleton JA, Lu P, et al.: Influenza vaccination of healthcare workers in the United States, 1989–2002. Infect Control Hosp Epidemiol. 2006; 27(3): 257–65. PubMed Abstract \| Publisher Full Text\n\nMaiman LA, Becker MH: The health belief model: origins and correlates in psychological theory. Health Educ Behav. 1974; 2(4): 336–353. Reference Source\n\nGlanz K, Rimer BK, Lewis FM: Health behavior and health education: Theory, research, and practice. 2002; 3rd ed. San Francisco: Jossey-Bass. Reference Source\n\nBond L, Nolan T: Making sense of perceptions of risk of diseases and vaccinations: a qualitative study combining models of health beliefs, decision-making and risk perception. BMC Public Health. 2011; 11: 943. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nToronto CE, Mullaney SM: Registered nurses and influenza vaccination. An integrative review. AAOHN J. 2010; 58(11): 463–71. PubMed Abstract \| Publisher Full Text\n\nRehmani R, Memon JI: Knowledge, attitudes, and beliefs regarding influenza vaccination among healthcare workers in a Saudi hospital. Vaccine. 2010; 28(26): 4283–4287. PubMed Abstract \| Publisher Full Text\n\nHunt C, Arthur A: Student nurses’ reasons behind the decision to receive or decline influenza vaccine: a cross-sectional survey. Vaccine. 2012; 30(40): 5824–5829. PubMed Abstract \| Publisher Full Text\n\nKoharchik LS, Salman K, Hardy E, et al.: Influenza immunisation status among nursing students. J Infect Prev. 2012; 13(3): 84–87. Publisher Full Text\n\nBrandt C, Rabenau HF, Bornmann S, et al.: The impact of the 2009 influenza A(H1N1) pandemic on attitudes of healthcare workers toward seasonal influenza vaccination 2010/11. Euro Surveill. 2011; 16(17): 1–6. PubMed Abstract\n\nClark SJ, Cowan AE, Wortley PM: Influenza vaccination attitudes and practices among US registered nurses. Am J Infect Control. 2009; 37(7): 551–556. PubMed Abstract \| Publisher Full Text\n\nHothersall EJ, de Bellis-Ayres S, Jordan R: Factors associated with uptake of pandemic influenza vaccine among general practitioners and practice nurses in Shropshire, UK. Prim Care Respir J. 2012; 21(3): 302–307. PubMed Abstract \| Publisher Full Text\n\nMehta M, Pastor CA, Shah B: Achieving optimal influenza vaccination rates: a survey-based study of healthcare workers in an urban hospital. J Hosp Infection. 2008; 70(1): 76–79. PubMed Abstract \| Publisher Full Text\n\nFalomir-Pichastor JM, Toscani L, Despointes SH: Determinants of flu vaccination among nurses: the effects of group identification and professional responsibility. Appl Psychol. 2009; 58(1): 42–58. Publisher Full Text\n\nOfstead CL, Tucker SJ, Bebe TJ, et al.: Influenza vaccination among registered nurses: Information receipt, knowledge, and decision-making at an institution with a multifaceted educational program. Infect Control Hosp Epidemiol. 2008; 29(2): 99–106. PubMed Abstract \| Publisher Full Text\n\nNovick MR: The axioms and principal results of classical test theory. J Math Psych. 1966; 3(1): 1–18. Publisher Full Text\n\nBean SJ, Catania JA: Vaccine perceptions among Oregon health care providers. Qual Health Res. 2013; 23(9): 1251–1266. PubMed Abstract \| Publisher Full Text\n\nSunstein CR: Moral heuristics. Behav Brain Sci. 2005; 28(4): 531–73. PubMed Abstract \| Publisher Full Text\n\nCochrane LJ, Olson CA, Murray S, et al.: Gaps between knowing and doing: understanding and assessing the barriers to optimal health care. J Contin Educ Health Prof. 2007; 27(2): 94–102. PubMed Abstract \| Publisher Full Text\n\nD’Souza C, Zyngier S, Robinson P, et al.: Health belief model: evaluating marketing promotion in a public vaccination program. J Nonprofit Public Sector Mark. 2011; 23(2): 134–157. Publisher Full Text\n\nRobertson L, O’Neill B, Wixom CW: Factors associated with observed safety belt use. J Health Soc Behav. 1972; 13(1): 18–24. Publisher Full Text\n\nCromby J: Beyond belief. J Health Psych. 2012; 17(7): 943–957. PubMed Abstract \| Publisher Full Text\n\nWhalen D, Molnar D, Milne F, et al.: Data Predicting Flu Vaccine Based on Experiences and Beliefs. figshare. 2014. Data Source" }	[ { "id": "6724", "date": "18 Nov 2014", "name": "M. Rashad Massoud", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAn interesting finding that even nurses are guided more by personal beliefs than medical knowledge in their personal health practices.Influenza virus contributes to morbidity and mortality via annual epidemics, and pandemics every 10-50 years. These epidemics can be moderated by mass vaccination and herd immunity. Nursing faculty are role models for their students, who are future nurses, as well as their patients. The authors used a cross-sectional survey based on the Health Belief Model to assess knowledge, attitudes and beliefs (KABs) on annual influenza vaccination of nursing faculty members, nursing students, and members of the public. The sample was recruited from three sources: 1) convenience sampling method of nursing faculty and students attending an annual research conference (226 surveys collected of 1000 distributed; 23%); 2) nursing students and faculty attending a health fair; 3) Community members chosen randomly. Logistic regression was performed to assess the extent to which influential personal experiences, vaccine beliefs index and vaccine knowledge index were predictive of flu vaccine receipt. Interestingly, positive experiences had a greater effect on influencing behavior than negative experiences. Response rate was low (23%) or note reported, and there was a small sample size: 29 nursing faculty, 197 nursing students, 152 community members. Therefore, the power of the analysis was low (0.63). It was also not clear how the community members were recruited. Due to the small sample size, and selection bias issues in the convenience sampling method, the generalizability of the study is limited and should be interpreted with caution. It is also not clear how the recommendations in the Discussion (e.g. social marketing campaign) were decided upon, as the effectiveness of such an approach is not clear, especially in comparison to other potential approaches. Overall, the underlying premise and theory is interesting and merits further study.", "responses": [] } ]	2	https://f1000research.com/articles/3-224
https://f1000research.com/articles/3-263/v1	31 Oct 14	{ "type": "Research Note", "title": "Cash, carrots, and sticks: Open Access incentives for researchers", "authors": [ "Joseph Kraus" ], "abstract": "Each country, scholarly field and institution has developed responses to new scholarly communication systems, and those policies and responses influence the behavior of the scholars within those systems. Over the last couple of years, policy makers and stakeholders in the United Kingdom have thoroughly discussed open access issues. In July 2012, the Finch Report and the Research Councils UK (RCUK) Policy on open access were published. The RCUK, one of the major funding bodies for research in the UK, announced the availability of a new funding mechanism to help researchers at member institutions transition their sponsored work to open access sources. Because the author is more familiar with the scholarly communication situation in the United States, the author interviewed 16 people with international perspectives on scholarly communication issues. This article provides an overview of the discussions with those individuals.", "keywords": [ "With the advent of the Internet", "scholarly communication has undergone many changes", "and it will continue to change in the future. There are many technological innovations that allow for greater and faster sharing of information and knowledge throughout the world. However", "even with technological advances", "the peer-review system is still a slow process (Roberts", "1999", "Smith", "2006). The tenure and promotion system allows (or even encourages) scholars to be conservative (Murray-Rust", "2008) during this time of change (Cavanagh", "2012). Scholars are rewarded when they publish articles and books with traditional and well-known publishers. The researchers may not be encouraged to submit works to new and different publishing outlets (Rohe", "1998). Hence", "some institutions and scholarly fields can be slow to change course when new publishing options become available." ], "content": "Introduction\n\nWith the advent of the Internet, scholarly communication has undergone many changes, and it will continue to change in the future. There are many technological innovations that allow for greater and faster sharing of information and knowledge throughout the world. However, even with technological advances, the peer-review system is still a slow process (Roberts, 1999; Smith, 2006). The tenure and promotion system allows (or even encourages) scholars to be conservative (Murray-Rust, 2008) during this time of change (Cavanagh, 2012). Scholars are rewarded when they publish articles and books with traditional and well-known publishers. The researchers may not be encouraged to submit works to new and different publishing outlets (Rohe, 1998). Hence, some institutions and scholarly fields can be slow to change course when new publishing options become available.\n\nSince the author works at a medium-sized private university in the United States, he is most familiar with the financial and social incentives for researchers in the United States. After reading discussions concerning the proposed policy changes for research funding in the United Kingdom, he desired to learn more about the social and financial shifts occurred there. While much could be learned by reading many of blog posts, reports, news sources, and other content that cover higher education and open access policy in the United Kingdom, the author sought to discuss the matter directly with open access advocates who know more about the situation in the UK. Thus, the author interviewed 16 different individuals who have a greater understanding of the situation in the UK. Ten of the sixteen interviewees were based in the UK or Europe at the time of the interviews. Two different interviewees mentioned the “carrot and stick” approach as an incentive method for changing behavior. In addition to financial rewards (cash) for scholars and researchers, there are a number of other positive (carrots) and negative (sticks) reinforcement methods to encourage greater sharing of research.\n\nThere are many issues at play during this time of change. The interviewees were asked how they viewed the policy changes occurring within the UK government, and discussed some other aspects of the scholarly communication system. Many scholars are concerned about the rise of the open access megajournals, such as PLOS ONE. Scholars are investigating alternative ways to measure the use, citation and interaction of content with their readers. “Altmetrics” is becoming the term of art (Lapinski et al., 2013). With new scholarly communication mechanisms, scholars are reinvestigating the issues of journal prestige, the use of impact factors, and the role of tenure and promotion committees. Scholars join scholarly societies, and are concerned about the effect of new publication models on those societies. Lastly, the UK uses the Research Excellence Framework (REF) to assess the quality of research within their higher education institutions (http://www.ref.ac.uk/). Many scholars in the UK are concerned about how the changing policies will affect the assessment process.\n\n\nOverview of Open Access developments\n\nFor those not familiar with open access principles in general, one should consult the books by Dr. Peter Suber (2012) and Crawford (2011). More recently, Vincent & Chris (2013) edited the book Debating Open Access for the British Academy for the Humanities and Social Sciences. These three sources provide good overviews of the topic.\n\nSpecific to the situation in the UK, Dr. Suber wrote about the movements toward open access in the UK and Europe in the SPARC Open Access Newsletter (2012). However, this covers many of the events through September of 2012, and there have been additional changes to documents and policies since then.\n\n\nThe Finch Report\n\nIn March 2011, the Honorable David Willetts MP, UK Minister for Universities and Science, recommended that an independent working group be formed to investigate “a programme of action and make recommendations to government, research funders, publishers and other interested parties on how access to research ﬁndings and outcomes can be broadened for key audiences such as researchers, policy makers and the general public” (http://www.researchinfonet.org/publish/finch/wg/).\n\nIn October 2011, the Working Group on Expanding Access to Published Research Findings was set up, and it was chaired by Dame Janet Finch DBE, Professor of Sociology at Manchester University and independent co-Chair of the Council for Science and Technology.\n\nThe report from the working group was released on June 18th, 2012, and it had not been modified since its release. The full title of the report is Accessibility, Sustainability, Excellence: How to Expand Access to Research Publications. Most researchers refer to this report simply as the Finch Report (2012).\n\nOn July 11, 2012, SPARC Europe responded to the Finch Report (Wellander, 2012).\n\nOn July 16, 2012, the UK government announced that it accepted the findings of the Finch Group. (http://www.researchinfonet.org/government-accepts-finch-proposals/).\n\n\nResearch Councils United Kingdom (RCUK) Open Access policy\n\nOn July 16th, 2012, The Research Councils United Kingdom (RCUK) simultaneously published their open access policy document. After the initial policy was published on July 16, 2012, there were a number of times when the policy was clarified or modified to better meet the needs of the scholars. The most recent policy statement was updated on April 8, 2013 (Research Councils UK, 2013). Dr. Mark Thorley, Chair of the RCUK Research Outputs Network, was the lead person from the RCUK who published some of the clarifications to the policy.\n\n\nInterview questions: summary of responses\n\nDuring October, November, and December of 2012, the author interviewed 16 researchers, scientists and scholars who were knowledgeable of the situation in the UK and Europe. Ten of the 16 interviewees were based in the UK or Europe at the time of the interviews. The other six are familiar with the scholarly communication system. In addition to questions that were specific to the Finch Report and the RCUK policy, the author also asked the interviewees about other aspects of scholarly communication. These included the topics of megajournals, journal prestige, society publishing, altmetrics, and the Research Excellence Framework.\n\n\nGreen and gold Open Access methods\n\n1) The Finch Report and the RCUK report recently came out. These reports have taken stances concerning green and gold Open Access in the UK. What are your thoughts on the issue of green vs. gold open access policies?\n\nThe respondents had a mix of recommendations. While it is clear that many scientists and researchers would prefer that the final published version be the open access version of record, there are many proponents of institutional repositories who would like to see their work get more use and status. The Finch report indicated that the usefulness of institutional repositories was being downgraded. However, it was noted that RCUK policy has wider latitude. While the Finch Report recommended a gold open access route, the RCUK could take a stance that recommends both green and gold open access methods. There was one individual who recommended a third method. This person noted that libraries could be much more involved in publishing research works.\n\n\nMegajournals\n\nSolomon & Björk (2012) describe megajournals as sources that have “very broad scopes. These journals have quick submission-to-publication times and only screen for scientific reliability, leaving it to the readers rather than the reviewers to judge the relevance”.\n\nNorman (2012) noted that these journals shared a number of features, such as sound science, academic editors, automated and scalable workflows, fast turnaround time, APCs around GBP £ 1,000, post-publication promotion, and article-level metrics.\n\nSome scholars are starting to see that the title of the journal that they publish in is becoming less and less important. Researchers are seeing less of a connection between past journal title prestige and the possible future impact through open access readership (Lozano et al., 2012). Thus, researchers are willing to publish their articles in broad-based open access journals that do not have niche titles.\n\n2) PLOS ONE is a well-known large open access journal that covers a broad range of disciplines. Because it has been deemed successful, other publishers have also proposed or started similar journals. What is your opinion of this new type of publication outlet?\n\nMost of the interviewees saw the rise of new magajournals as a positive development. There is space in the scholarly publishing industry to support many megajournals as well as niche journals. In the future, the megajournal will simply be called a journal that happens to have a broad scope and appeal.\n\n\n“Move the prestige to Open Access”\n\nAs stated in the Finch Report, moving prestige to open access will entail a change of culture.\n\nSection 4 (What needs to be done, page 7) begins as follows:\n\nImplementing our recommendations will require changes in policy and practice by all stakeholders. More broadly, what we propose implies cultural change: a fundamental shift in how research is published and disseminated.\n\nDr. Michael Taylor (2012) wrote up some thoughts about the Finch Report, and he noted that:\n\nCultural change is exactly what’s needed — not just in how research is published, as noted in the report, but even more importantly in how it’s evaluated. In particular, we’re going to have to stop assessing research by what journal it’s published in, and start looking at the value of the actual research.\n\nHarvard University had noted in this report, “Faculty Advisory Council Memorandum on Journal Pricing”, (http://isites.harvard.edu/icb/icb.do?keyword=k77982&tabgroupid=icb.tabgroup143448) that researchers and authors from Harvard should “consider submitting articles to open-access journals, or to ones that have reasonable, sustainable subscription costs; move prestige to open access”.\n\nThe concept of asking faculties to move their prestige to open access is interesting, but it entails a cultural shift within hundreds of universities and departments. Because of local circumstances, some universities and colleges may have a harder time requesting their faculty to move their prestige. After the initial question, the author asked for practical advice on how to recommend this path to faculty at his institution.\n\n3) Harvard University has recommended to their faculty to “consider submitting articles to open-access journals, or to ones that have reasonable, sustainable subscription costs; move prestige to open access” (http://isites.harvard.edu/icb/icb.do?keyword=k77982&tabgroupid=icb.tabgroup143448). The concept of “moving prestige to open access” is an interesting statement to the Harvard faculty authors and researchers. What do you think of this statement?\n\nSome of the interviewees said that the pressure to move prestige to open access sources should continue to come from funding agencies, since those organizations would like to see the widest uptake of the research that they had funded. Other interviewees noted that open access proponents should discuss the issue with lower level researchers and students. Once the scholarly publication system is explained to younger scholars, it will be obvious to them to publish their work in open access sources. However, younger scholars have to live in the scholarly publishing world as it currently exists. Even if several educational institutions issued a statement similar to Harvard University, this would reach a small minority of researchers. This is a collective action problem. Not only do researchers at many large educational institutions have to believe they can move their authority to open access, they have to act on that.\n\n\nThe effect of Open Access on societies\n\nThere are quite a number of viewpoints concerning how the changing scholarly communication ecosystem is going to affect society publishing (Jump, 2013; Shieber, 2013; Morris & Thorn, 2009). In particular, Dr. Neylon (2012) addressed this topic in a blog post:\n\nWith major governments signalling a shift to open access it seems like a good time to be asking which organisations in the scholarly communications space will survive the transition. It is likely that the major current publishers will survive, although relative market share and focus is likely to change. But the biggest challenges are faced by small to medium scholarly societies that depend on journal income for their current viability.\n\nHowever, it should be noted that Dr. Neylon works for PLOS as their Advocacy Director.\n\n4) University presses and many societies are concerned about how the open access movement will affect their financial bottom line. What concerns do you have about open access and society publications?\n\nMany of the respondents expressed a concern for the long term survival of scholarly societies. Historically, scholarly societies have published the highest quality and the most cost-effective research journals. Some respondents recommend that the services that societies provide to scholars be unbundled from their publishing activities. For example, the funding for conference activities should be separated from publishing activities. Several people recommended that societies use alternative funding models, such as a PeerJ model or a SCOAP3 model. Others recommended that more societies could collaborate to save on costs, and use the same publishing platform. Another person recommended that societies could take advantage of open source software such as OJS as a publishing platform. Learned societies have adapted to changes in the past, and they will continue to do so. In the future, the most successful societies will focus on their mission to disseminate research in the most cost-effective way.\n\n\nThe rise of Altmetrics\n\nAltmetrics is a new field of endeavor, and it has been widely discussed in the literature. Several articles (Adie & Roe, 2013; Eysenbach, 2011; Mounce, 2013; Priem et al., 2012; Baynes, 2013) will provide background reading if needed.\n\n5) Altmetrics is gathering steam as an additional method for faculty to determine the impact of their work (http://altmetrics.org). Do you plan to take advantage of these data for either your work, or for the benefit of your institution or department?\n\nMost of the interviewees see a connection between the measurement of individual research items and researcher impact and altmetrics. All of the interviewees plan to keep up with the research in altmetric systems. Some of the interviewees were concerned with the use of social media mentions as part of an altmetric score. Some also had concerns with the possibility of researchers gaming the altmetric system. However, most understand the limitations of article citations as the only measure of scholarship value. Several interviewees mentioned that researchers publish work that is not in the form of a journal article. Many researchers write computer code, publish data, present conference papers, and more. This non-article work is difficult to measure using traditional citation metrics. With additional data points beyond Thomson Reuters journal Impact Factors, researchers will be able to document readership and the use of their work that goes beyond citation counts. By taking advantage of altmetric measurement systems, scholars who publish in non-elite and nontraditional sources can demonstrate their value as a scholar. In other words, scholars can publish strong work in less established sources and show that the work had an impact.\n\n\nImpact factors, the Research Excellence Framework (REF), and the Wellcome Trust\n\nThe Impact Factor that is published by Thomson Reuters has been widely used in scholarly circles as a proxy for journal prestige. The higher the number, the higher the perceived prestige of a journal. There is a great amount of research surrounding the manipulation (Yu & Wang, 2007) and use of the Impact Factor number (Garfield, 2006). During May 2013, about 150 researchers and 75 organizations (Basken, 2013) issued the “San Francisco Declaration on Research Assessment (DORA)”. The signers of the document recognized “the need to improve the ways in which the outputs of scientific research are evaluated” (http://am.ascb.org/dora/).\n\nIn the UK, there are two major organizations that do not take the Impact Factor (or the perceived prestige of a journal title) into account when determining the quality of published research.\n\nThe Wellcome Trust “affirms the principle that it is the intrinsic merit of the work, and not the title of the journal in which an author’s work is published, that should be considered in making funding decisions” http://www.wellcome.ac.uk/About-us/Policy/Spotlight-issues/Open-access/Policy/index.htm.\n\nTheir FAQ section of the REF notes that “No sub-panel will make any use of journal impact factors, rankings, lists or the perceived standing of publishers in assessing the quality of research outputs. An underpinning principle of the REF is that all types of research and all forms of research outputs across all disciplines shall be assessed on a fair and equal basis” http://www.ref.ac.uk/faq/all/.\n\n6) The Research Excellence Framework (REF) in the UK notes: “No sub-panel will make any use of journal impact factors, rankings, lists or the perceived standing of publishers in assessing the quality of research outputs” (http://www.ref.ac.uk/faq/all/). While this is a valid statement for UK based research evaluation, it would be impossible to get a majority of academic tenure and promotion committees throughout the United States to agree to a similar statement in the near future. Since the UK has the REF, and the US does not, how much is this holding back the US from adopting greater OA policies at various institutions?\n\nSome of the respondents felt that the REF works for a country the size of the UK, but it wouldn’t work for the United States. Some of the interviewees noted that the REF statement concerning journal impact factors and rankings were not believed by many researchers in the UK, so a statement by United States officials may not be believed as well. If some United States educational institutions were to adopt similar policies, it may not change the perceived value of elite status journals by researchers in those fields.\n\nFunder policies and financial incentives were mentioned by some of the interviewees. Researchers will follow the money. If funders want researchers to publish using open access methods, then researchers will do what the funders demand to receive that funding. In short, the lack of policy statements by US educational institutions concerning the use of “journal impact factors, rankings, lists or the perceived standing of publishers” is probably not holding the US from adopting more OA policies. This not to say that adopting a policy along these lines would not be taken as a positive step, but there are other cultural factors that are limiting the number of open access policies at US educational institutions.\n\n\nConclusion\n\nThe author saw that the UK Government was addressing the open access publishing problem head on, and he desired to learn more about how local researchers felt about the discussions surrounding open access and other scholarly communications issues. After meeting with 16 scholars and researchers, he has come away with a more nuanced understanding of the open access landscape.\n\nThere are many ways for researchers to disseminate the results of their research. Some open access advocates favor the institutional repository method, and others favor a gold open access method through publishers. This presents a false dichotomy. There is no “One Best Way” in the path of greater Open Access. Some researchers may wish to summarize their research and post it to a blog. Some researchers may want to synthesize their findings and put presentation slides onto SlideShare. Other researchers might wish to place drafts of manuscripts onto a personal website, instead of using an institutional repository. Different scholars in different disciplines will have different ways of sharing their research with the world. As a librarian, I can let researchers know about the advantages and disadvantages of various open access methods, but in the end, the researcher is the one who will decide what is best for them and their research.\n\nThere is not much controversy over the rise of megajournals. Many interviewees see it as a natural step in the progression and growth of scholarly journals as they transition to the Internet.\n\nHarvard University may recommend to their faculty that they move their authoritative prestige to open access, but this policy has not caught the attention of many other administrators at institutions in the UK nor in the US. The interviewees noted that while this is a good idea, scholars must consider other cultural and financial issues as they move their work to open access sources.\n\nThere are many sizes and types of learned societies, and they have varying levels and sizes of publishing programs. For the most part, librarians support society publishing because they publish some of the most cost effective journals. But, many of the smaller society publishers are either nonprofit or they have very narrow margins. Thus, they may not be in a position where they can experiment with different funding and publishing models. During the transition to open access publishing, societies will need to carefully examine their mission and focus on meeting the information needs of their members and authors.\n\nAltmetrics is poised to provide a new way for scholars to measure their impact within their fields. Since there are new communication methods popping up all the time on the Internet, scholars will have new ways of reaching different audiences. Altmetrics will help the scholars demonstrate the value of new communication methods to administrators at their institutions.\n\nThe REF may state that “No sub-panel will make any use of journal impact factors, rankings, lists or the perceived standing of publishers in assessing the quality of research outputs”, but that doesn’t mean that most people believe it. There are many scholars who still believe that an article that is published in an elite journal will receive higher status because it is published in a high prestige journal. The perceived prestige and status of different journals and publishers can last for a long time.\n\nTo close the article, some insightful closing comments from the interviewees are provided.\n\n“We get the impression that people in the US want open access, but they are not prepared to pay for it”. [The US say] ‘Let’s go for green’.\n\nSome of the supporters of green OA “have a strong anti-publisher rhetoric, and this does worry me at times”. “The journals are a key part of the quality step”.\n\n“We want a transparent APC market, so we can start to create a functioning market in scholarly communication”. “Currently, it is a free market for the authors”. “They are not aware of the tough choices that libraries have to make”. Researchers should “take more responsibility for the dissemination of their research and understand the costs involved in that”. Scholars are also learning that “where they choose to place their publication can limit the access to that research”.\n\n“We are close to or at least within reach of several of those tipping points that we have been looking at for quite some time”. “Things will run very fast, and probably out of our control over the next couple of years. How do we prepare for the avalanche, as it were?”\n\n“The fundamental problem at the heart of the crisis in scholarly communication is the way in which the peer-reviewed paper has become the main currency for tenure and promotion. This has led to a gross inflation of unmemorable, unreadable, irrelevant and/or pointless papers. It has also led to fraud, to shoddy science and to a commensurable rise in retractions and scandals. Finally, it has led to a plague of for-profit publishers whose primary concern is not scholarly communication, but maximising profit”. “In brief, OA is too often viewed as a solution to the problems of scholarly communication, but it is becoming increasingly clear that it could in fact exacerbate these problems, particularly as the research community becomes more and more focused on pay-to-publish gold OA”.\n\n“The policies and procedures that are implemented today concerning new communication systems can have an effect on that future”.\n\n“My recommendation is library support for scholar-led publishing as the most cost-effective solution for the future”.\n\n“The real surprises are still out there”. “Open access is just starting” to get a foothold in the marketplace. “It is possible to make good money off of open access”. “Entrepreneurship is important. Entrepreneurs need the freedom to fail cheaply”. If we “increase the sample size, then we can increase the numbers of successes”.\n\n“I don’t think I know where we are going. We are in a period where there is going to be a lot of experiments, some of which are not going to work, some of which may partially work, but that will teach us things”. “I think that there are dramatic changes coming to the scholarly communications system”. “The main thing I think we need is a willingness to try different things, a willingness to fail, and an attitude of humility. See what we can learn as we go along”.\n\n\nConsent\n\nWritten informed consent for publication of these results has been obtained by each interviewee. The text in the interview sections do not indicate the respondent in order to conform to the IRB request.", "appendix": "Competing interests\n\n\n\nThe author is an open access advocate, and he is involved in the editing and publishing of two open access journals. They are Collaborative Librarianship and the Journal of Creative Library Practice. These two journals are in no way affiliated with F1000 Research.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work. The author received a one-quarter sabbatical in 2012 from the University of Denver to perform this research.\n\n\nAcknowledgements\n\nI would like to thank all participants for responding to the interview questions.\n\n\nReferences\n\nAdie E, Roe W: Altmetric: enriching scholarly content with article-level discussion and metrics. Learn Publ. 2013; 26(1): 11–17. Publisher Full Text\n\nBasken P: Researchers and scientific groups make new push against impact factors. Chron High Educ. 2013. Reference Source\n\nBaynes G: Key Issue - Scientometrics, bibliometrics, altmetrics: Some introductory advice for the lost and bemused. Insights: the UKSG journal. 2012; 25(3): 311–315. Publisher Full Text\n\nCavanagh S: Living in a digital world: Rethinking peer review, collaboration, and Open Access. ABO: Interact J Women Arts. 2012; 2(1): 1640–1830. Publisher Full Text\n\nCrawford W: Open Access: What You Need to Know Now. Chicago: American Library Association, 2011. Reference Source\n\nEysenbach G: Can tweets predict citations? Metrics of social impact based on Twitter and correlation with traditional metrics of scientific impact. J Med Internet Res. 2011; 13(4): e123. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFinch J; The Working Group on Expanding Access to Published Research Findings: Accessibility, sustainability, excellence: how to expand access to research publications. 2012. Reference Source\n\nGarfield E: The history and meaning of the journal impact factor. JAMA. 2006; 295(1): 90–93. PubMed Abstract \| Publisher Full Text\n\nJump P: Open access will cause problems for learned societies' journals, accepts Finch. Times Higher Education. 2013. Reference Source\n\nLapinski S, Piwowar H, Priem J: Riding the crest of the altmetrics wave. Coll Res Libr News. 2013; 74(6): 292–300. Reference Source\n\nLozano G, Larivière V, Gingras Y: The weakening relationship between the impact factor and papers' citations in the digital age. J Am Soc Inf Sci Technol. 2012; 63(11): 2140–2145. Publisher Full Text\n\nMorris S, Thorn S: Learned society members and open access. Learn Publ. 2009; 22(3): 221–239. Publisher Full Text\n\nMounce R: Open Access and Altmetrics: Distinct but Complementary. Bull Assoc Info Sci Tech. 2013; 39(4): 14–17. Publisher Full Text\n\nMurray-Rust P: Open Access in chemistry – thoughts for Thursday. Petermr's blog: A Scientist and the Web. 2008. Reference Source\n\nNeylon C: The challenge for scholarly societies. Science in the Open. 2012. Reference Source\n\nNorman F: Megajournals. Trading Knowledge. 2012. Reference Source\n\nPriem J, Piwowar HA, Hemminger BM: Altmetrics in the wild: Using social media to explore scholarly impact. 2012; arXiv.. Reference Source\n\nResearch Councils UK: RCUK Policy on Open Access and Supporting Guidance. 2013. Reference Source\n\nRoberts P: Scholarly publishing, peer review and the Internet. First Monday. 1999; 4(4). Publisher Full Text\n\nRohe TA: How does electronic publishing affect the scholarly communication process? J Electron Publ. 1998; 3(3). Publisher Full Text\n\nShieber S: Why open access is better for scholarly societies. The Occasional Pamphlet. 2013. Reference Source\n\nSmith R: Peer review: A flawed process at the heart of science and journals. J R Soc Med. 2006; 99(4): 178–182. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSolomon DJ, Björk B: A study of open access journals using article processing charges. J Am Soc Inf Sci Technol. 2012; 63(8): 1485–1495.Publisher Full Text\n\nSuber P: Open Access. Cambridge, MA: MIT Press, 2012. Reference Source\n\nSuber P: Tectonic movements toward OA in the UK and Europe. SPARC Open Access Newsletter. 2012; (165). Reference Source\n\nTaylor M: Thoughts on the Finch Report, part 2. Sauropod Vertebra Picture of the Week. 2012. Reference Source\n\nVincent N, Chris W, Rita G, et al.: Debating Open Access. London: The British Academy. 2013. Reference Source\n\nWellander J: SPARC Europe’s response to the Finch Report. SPARC Europe. 2012. Reference Source\n\nYu G, Wang L: The self-cited rate of scientific journals and the manipulation of their impact factors. Scientometrics. 2007; 73(3): 321–330. Publisher Full Text\n\n\nAppendix 1\n\n1) The Finch report and the RCUK report recently came out. These reports have taken stances concerning green and gold open access in the UK. What are your thoughts on the issue of green vs gold open access policies?\n\n2) PLOS ONE is a well-known large open access journal that covers a broad range of disciplines. Because it has been deemed successful, other publishers have also proposed or started similar journals. What is your opinion of this new type of publication outlet?\n\n3) Harvard University has recommended to their faculty to “consider submitting articles to open-access journals, or to ones that have reasonable, sustainable subscription costs; move prestige to open access” (http://isites.harvard.edu/icb/icb.do?keyword=k77982&tabgroupid=icb.tabgroup143448). The concept of “moving prestige to open access” is an interesting statement to the Harvard faculty authors and researchers. What do you think of this statement?\n\n4) University presses and many societies are concerned about how the open access movement will affect their financial bottom line. What concerns do you have about open access and society publications?\n\n5) AltMetrics is gathering steam as an additional method for faculty to determine the impact of their work (http://altmetrics.org). Do you plan to take advantage of this data for either your work, or for the benefit of your institution or department?\n\n6) The Research Excellence Framework (REF) in the UK notes: “No sub-panel will make any use of journal impact factors, rankings, lists or the perceived standing of publishers in assessing the quality of research outputs” (http://www.ref.ac.uk/faq/all/). While this is a valid statement for UK based research evaluation, it would be impossible to get a majority of academic tenure and promotion committees throughout the United States to agree to a similar statement in the near future. Since the UK has the REF, and the US does not, how much is this holding back the US from adopting greater OA policies at various institutions?\n\n7) Is there anything else you would like to say concerning open access publishing?" }	[ { "id": "6619", "date": "21 Nov 2014", "name": "Micah Vandegrift", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article provides a helpful overview of the state of open access in the UK. Coming from a similar perspective as the author (working in scholarly communication at a university in the US), the focus of this piece was helpful to consolidate what often sounds like a lot of momentum toward open access from the UK. Between the title and the abstract, I was expecting a different paper than was presented; for example, perhaps a more appropriate title would be “Cash, carrots and sticks: A Survey of Recent Developments in Open Access in the United Kingdom.” Extrapolating from the abstract, I had hoped the article would begin with the UK, but also include the “international perspectives on scholarly communication issues” from Australia, Latin America and Asia. The organization of the paper could be improved by grouping sections a little differently. I see 3 major sections in the work, Intro/background context, Current Topics in ScholComm, and Survey/Results. “The Overview of OA developments” could easily be appended to the Introduction section, followed by a top level heading introducing all the “current topics” around which the author constructed the survey: The Finch Report, RCUK, Green vs. Gold, Megajournals, prestige in open access, open access and scholarly societies, and altmetrics vs impact factor. I agree completely with the author that these topics are absolutely at the core of the current scholcomm conversation. Finally, a new section could follow titled Survey, Methodology and Analysis of Responses wherein the author synthesizes the survey responses referring to the context and current issues as necessary. Then, of course, close the paper with a conclusion. Reorganizing the paper would go a long way toward making its findings more digestible and impactful. In many of the responses to the survey questions, the author uses the word “many” or “most.” Every time I read that I wondered, does that mean 50%, 75% or 90%? Even with the small sample size numerically representing the responses would be helpful. Also, including the question in the text of the article as it was written in the survey instrument makes the article read choppily. I’d prefer to read the question written into prose with the responses; for example, “When asked of their opinion on the ride and impact of megajournals like PLoSOne, 15/16 respondents agreed that this type of publishing will grow in popularity in the future.” The conclusion could be stronger by changing its focus from detailing each issue (green v. gold, megajournals, societies, etc.) to offering the author’s perspective on why or why not the UK seems to be more forward-thinking in terms of open access policy at the governmental level. What is the author’s “more nuanced understanding of the OA landscape”? Further, I’d love for the author to propose how this study might be replicated, extended, and expanded to a broader international scope. My own personal perspective is that a global view of how and why open access has real affects is absolutely necessary for the growth of this sub-field of librarianship. The promise of this article to provide an “international perspective” is exactly what we need. I’ll push the author and those reading this piece to work toward that goal.", "responses": [ { "c_id": "1208", "date": "06 Feb 2015", "name": "Joseph Kraus", "role": "Author Response", "response": "I appreciate your comments and the recommendations you provided. I will take your response into consideration for the updated version of the article." } ] }, { "id": "6615", "date": "27 Nov 2014", "name": "John Dupuis", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSuitability of TitleThe title should probably mention that the article was focused on the situation in the UK.Does Abstract Accurately Summarize ArticleYes, but may have to change if the article changes.ReviewMost of my concerns with this article has to do with the demographics of the interviewees.The author does not clearly specify how the interviewees were selected. For example, how was the pool of potential interviewees created? Within that pool, how were the actual interviewees selected? Was it random or where there criteria involved?Even taking into account the confidentiality of the interviews, it may have been possible to be more specific about demographics. Were all the interviewees scientists or were some publishers or librarians or others? How do those categories break down? What was the rationale for selecting non-UK interviewees for learning more about the situation in the UK? Were the interviews conducted by phone, email or in person?Some other issues:In the summary of responses, while I appreciate that the various responses are summarized and digested, I also think there are numerous opportunities to be more specific in how the responses are coded. For example, the words such as “many,” “most,” and “some” are used frequently. The message of the article would benefit from more exactness. Is many 5 or 6? Is most 9 or 15? Some summaries could easily have benefited from charts or graphs. Related, the text of the interviews is extremely rich. Even given that the article is a Research Note, the summaries of responses could have reflected that richness better. I appreciate that the raw text of the interview responses is available to us. However, it would have been nice to code the responses in such a way that readers could track the various responses by interviewee. For example, S1, S2, etc, for Subject 1, Subject 2, etc. The material in the conclusions was perhaps a little too much on the summary side and could have benefited a little more in terms of discussion and perhaps what further research is needed.", "responses": [ { "c_id": "1207", "date": "06 Feb 2015", "name": "Joseph Kraus", "role": "Author Response", "response": "Thank you very much for the comments. I will take your response into consideration for the updated version of the article." } ] } ]	1	https://f1000research.com/articles/3-263
https://f1000research.com/articles/3-261/v1	31 Oct 14	{ "type": "Opinion Article", "title": "Collaboration for rare disease drug discovery research", "authors": [ "Nadia K. Litterman", "Michele Rhee", "David C. Swinney", "Sean Ekins", "Michele Rhee", "David C. Swinney" ], "abstract": "Rare disease research has reached a tipping point, with the confluence of scientific and technologic developments that if appropriately harnessed, could lead to key breakthroughs and treatments for this set of devastating disorders. Industry-wide trends have revealed that the traditional drug discovery research and development (R&D) model is no longer viable, and drug companies are evolving their approach. Rather than only pursue blockbuster therapeutics for heterogeneous, common diseases, drug companies have increasingly begun to shift their focus to rare diseases. In academia, advances in genetics analyses and disease mechanisms have allowed scientific understanding to mature, but the lack of funding and translational capability severely limits the rare disease research that leads to clinical trials. Simultaneously, there is a movement towards increased research collaboration, more data sharing, and heightened engagement and active involvement by patients, advocates, and foundations. The growth in networks and social networking tools presents an opportunity to help reach other patients but also find researchers and build collaborations. The growth of collaborative software that can enable researchers to share their data could also enable rare disease patients and foundations to manage their portfolio of funded projects for developing new therapeutics and suggest drug repurposing opportunities. Still there are many thousands of diseases without treatments and with only fragmented research efforts. We will describe some recent progress in several rare diseases used as examples and propose how collaborations could be facilitated. We propose that the development of a center of excellence that integrates and shares informatics resources for rare diseases sponsored by all of the stakeholders would help foster these initiatives.", "keywords": [ "rare disease", "patient advocacy", "drug discovery", "Twitter" ], "content": "Introduction\n\nAlthough each rare disease affects less than 200,000 individuals in the United States, in aggregate, rare diseases affect 6–7% of the population1. As less than 10% of these patients can be presently treated, this remains a very large unmet medical need2. According to the National Organization for Rare Disorders (NORD), there are only 250 treatments for the nearly 7,000 rare disorders, impacting nearly 30 million Americans. Eighty percent of these diseases have a genetic origin1,3. Most rare diseases are caused by mutations in a single gene, such as an enzyme deficiency (like α-galactosidase A in Fabry’s Disease). Other diseases, such as Charcot-Marie-Tooth (CMT), have multiple genetic causes4. In either case, the knowledge of the genetic basis of the disease can be quite illuminating and lead to therapeutic development efforts3.\n\nIn metabolic disorders such as the lysosomal storage diseases, the development of biologics such as enzyme replacement therapy has been quite fruitful. In rare cancers, the knowledge of mutated kinases has led to the development of specific, potent small molecule inhibitors5,6. There are many diseases with approved drugs developed by a knowledge of genetics such as: myelofibrosis, non-small cell lung cancer (NSCLC), late stage melanoma, chronic myelogenous leukemia (CML), Gaucher's disease, Pompe's disease, hyperphenylalaninemia, Hunter syndrome, mucopolysaccharidosis (MPS) VI, MPS I, Fabry's disease, Type I tyrosinemia, hyperammonemia due to N-acetylglutamate synthase (NAGS) deficiency, cystic fibrosis, hereditary angioedema (HAE), cryopryin-associated periodic syndromes, and paroxysmal nocturnal hemoglobinuria3.\n\nDeveloping novel therapeutics is always a risky and difficult endeavor7,8, and the process of drug discovery for rare diseases is marked by unique challenges. Rare diseases represent an example of the power of individualized therapies, but under the current paradigm the trade-off is that these are incredibly expensive9,10. So there is an urgent need to discover ways to develop therapies more cost-effectively11. Using our perspectives as rare disease researchers and patient advocates with experience of facilitating collaborations, we will use the diseases we have most knowledge of to outline the challenges that we see and offer some proposed solutions in this opinion article.\n\n\nGetting connected\n\nRare diseases, by definition, have very small numbers of patients (sometimes in the low tens to hundreds) that are often dispersed and disconnected globally. This is problematic for understanding the natural history of the disease, identifying the underlying mechanisms, and recruiting patients for clinical trials3. Before a therapy can be developed, the natural history needs to be understood so that the clinical trials can attempt to show a positive outcome with the disease in question. Despite the inherent difficulties, recent trends and technological developments, including in genomics, collaboration, and even social media, can be harnessed to the advantage of rare disease patients and researchers.\n\nThe connectivity and network-building enabled by the internet is especially important for rare disease patients and caregivers during diagnosis and treatment. Even well-informed individual physicians are unlikely to have experience with all given rare conditions, making diagnoses challenging. Despite the importance for support and knowledge sharing, it is extremely unlikely for rare disease patients to find one another through traditional methods like face to face networking, conferences, newspaper and magazine articles, etc., especially since the Health Insurance Portability and Accountability Act (HIPAA) privacy rules make it difficult12,13 to share information. Recent technological advancements have helped to reduce the barriers for doctors, caregivers, and patients to reach out to find one another. Social network sites such as Sermo14 and Doximity15 enable physicians to crowdsource a diagnosis. In addition, patients can find one another through websites and social media. For example, many rare disease groups set up public or private Facebook pages. Some are totally open and write regular blog posts to communicate their activities and goals, or what they have done to increase awareness, fund-raise, or look for a cure. In other cases they may be private sites for caregivers to use them to share their experiences. Both approaches enable families with very rare diseases to connect and then build momentum from there16,17. Many rare disease advocates are also users of Twitter as a tool to highlight articles of interest, promote their fundraising events, or just share their experiences (Table 1). Overall, this increased connectivity benefits patients, trying to identify the source of their symptoms and understand their recent diagnosis. In addition, such connected patient networks can also lead to key research breakthroughs, such as defining the genetic origin of the disease, understanding the natural history, defining biomarkers, and recruiting patients for clinical registries, natural history studies and clinical trials. A useful side effect of this social networking is to raise the overall level of awareness amongst the population that was previously not familiar with rare diseases.\n\n(Also see http://www.totalbiopharma.com/2013/07/01/top-50-social-media-influencers-orphan-drugs-rare-disease/ and http://moderators.rareconnect.org/social-media-case-studies/raredisease-patient-advocates-follow-these-25-twitter-accounts/).\n\n\nThe role of patient advocacy organizations\n\nHistorically, industrial sponsors of research and clinical trials see the rare disease space as riskier, and less profitable than more common diseases. The perception around profit has shifted as rare disease blockbuster drugs (such as Vertex’s Kalydeco™ for cystic fibrosis) make headlines. Still, key decision-makers within biopharmaceutical companies continue to be hesitant about pursuing rare disease indications due to the perceived risk. When the average drug costs $500 million–$1 billion (or more) and takes 15–20 years to develop7,8, companies want to reduce the likelihood of failure and increase the potential revenue as much as possible. Hence the rationale behind rare disease drugs that cost upwards of $100,000 per year. To be simplistic, the calculation considers whether the amount of money spent to get a drug to market will be less than the amount of money received in revenue over the lifetime of the patent exclusivity of the drug. The equation tends to come out on the wrong side for rare diseases, in large part because many of the attempts to get orphan drugs to market have failed, as evidenced by the lack of launched drugs. These failures increase the perception of riskiness of the space, which means that, at a minimum, companies are less likely to invest in programs focused on a rare disease.\n\nOne approach to solving this catch-22 problem is for patient advocacy groups to collaborate with academia, government, not-for profits and biopharmaceutical companies to increase incentives for investment in rare-disease-specific programs. Patient advocacy organizations play a role9,18,19 here because they can be uniquely strategic and creative to reduce risk, bring patients together for researchers and drive research forward.\n\nAt a base level, risk can be reduced if the cost of entering the disease area can be lowered, reducing the initial financial risk. When a company is deciding whether to pursue a specific indication for a disease, it is competing against others that may have existing pre-clinical models and clinical trial networks. The investment required is therefore often higher in rare diseases because the infrastructure does not exist. The barrier to entry can be mitigated through the support of and collaboration with disease-specific patient advocacy groups. Large organizations such as the Michael J. Fox Foundation for Parkinson’s Disease Research and the Cystic Fibrosis Foundation have used a modified venture philanthropy model. This allows them to invest in and provide research grants to biotechnology companies who have designated programs in their specific disease areas. These investments and grants become true collaborations over time as these large patient advocacy groups provide disease expertise, access to key opinion leaders and patients, and clinical trial recruitment support. The key word here is time. These collaborations can last over a decade. Obviously, however, not all rare disease groups have the financial resources to invest the funding required to significantly reduce risk which at this level is likely in the $10–100 millions range. Also, many groups are trying to discover therapies in a shorter time if possible. However, patient groups do not have to invest at this level to have an impact. Smaller investments in the low tens of thousands of dollars can have an impact in funding science9, providing the seed funding to develop an assay or animal model or even make a compound for testing. These efforts may more frequently be targeted to academia as long as the overhead costs can be kept to a minimum or avoided.\n\nBrain cancer offers an example of where a company was able to spread the financial risk across a number of patient advocacy groups. Tocagen applied for and received research grants from three major brain tumor patient advocacy groups: National Brain Tumor Society, Accelerate Brain Cancer Cure and American Brain Tumor Association. Although the financial support was helpful, the company also benefited by having the support of these patient advocacy organizations as it recruited for clinical trials. Typically, because of the small patient population numbers, clinical trial recruitment is a huge issue in rare disease clinical trials, but Tocagen has not suffered the traditional patient recruitment problems.\n\nOther collaborative approaches that do not require as much financial outlay can also be successful at encouraging companies to build or to further develop specific programs. These are typically more policy- and advocacy-based and are often areas in which only patient advocacy groups or other not-for-profits can lead. Existing policies that increase incentives to discover new medical entities for rare diseases include the Orphan Drug Act, pediatric priority review vouchers, and extended patent exclusivity upon the inclusion of a pediatric indication.\n\nPatient advocacy groups18 can also use their passion and experience to clarify some of the ambiguity in the regulatory environment for rare disease drug evaluations. Because there have been so few successes in the orphan drug space, drug companies have limited precedents to follow as they design their clinical trials and navigate the Food and Drug Administration (FDA). Typically, the FDA speaks to trial sponsors only within the context of a specific application, which is less of an issue when there have been recent or multiple approvals in a disease because the trial design and endpoints are clear. Companies will err on the conservative side and design trials with larger numbers of patients using endpoints that often take longer (for example, using overall survival instead of a surrogate endpoint).\n\nOne approach to clarifying this regulatory uncertainty is for the patient advocacy community to collaborate with key stakeholders to have open discussions about trial design and endpoints. For example, the Jumpstarting Brain Tumor Drug Discovery Coalition (comprised of the National Brain Tumor Society, the Society for Neuro-oncology, the Musella Foundation, and Accelerate Brain Cancer Cure) has hosted two workshops to discuss alternative and surrogate endpoints for clinical trials. FDA staff, trial sponsors, clinicians and scientists, clinical trial designers, and patients have all been actively involved in working with the patient advocacy coalition to identify endpoints and trial designs that will reduce the time and money required to run a clinical trial in brain cancer. Although the work is still ongoing, the FDA has pointed to this collaboration as an example for other patient advocacy groups to follow, and trial sponsors have enthusiastically participated. Indeed, this approach is being replicated by many rare diseases on an individual basis. Perhaps if all the different rare disease groups collaborated and had these discussions at one time, there could be synergistic effects in terms of prior experience, cost effectiveness and time-savings.\n\nThe previous examples of cross-sector collaboration all require either significant financial investment and/or labor outlays. Many rare disease patient advocacy organizations lack substantial funding resources, staff numbers, or even the experienced individuals needed to coordinate collaborations, which limits the options for collaborating with biopharmaceutical companies to encourage greater investment in rare diseases. However, there is an option that is less labor- or finance-intensive: advocating directly to and within the company to support the development of internal programs in rare diseases and providing disease expertise and information.\n\nThe National Brain Tumor Society, for example, has collaborated with two biotechnology companies to help them launch brain-cancer-specific programs internally. For both companies, there were a series of meetings and presentations by the patient advocacy group in order to educate the company about the following:\n\nUnmet need\n\nCurrent research landscape\n\nTreatment paradigm\n\nMarket potential/size\n\nOne of the companies is now collaborating with two of the National Brain Tumor Society’s funded researchers, and the other is funding a pilot program in brain cancer. The companies are leveraging their expertise in specific technology to adapt and optimize it for brain cancer treatment. These are pre-clinical programs, and it is anticipated that the relationships and collaborations between the National Brain Tumor Society and the biotechnology companies will continue throughout the development process. It is exciting to see novel technologies applied to a new area where the companies are taking the additional step of tailoring them to the unique needs of the biology of the disease and the patient population. Each company required a different approach, and all collaborations have to be structured to be sensitive to the needs and expectations of each. The passion and commitment of the patient advocacy groups involved were the key drivers in each case. These highly innovative programs have flourished because of the rare disease patient community. It is expected that continued innovation in identifying opportunities will allow further future successes.\n\nGiven the current funding environment for academic and startup researchers, it is interesting to note that there may be potential for a surge in rare disease interest and investment. It may seem counterintuitive, but we can look to the 2008 recession for the rationale. In a lean environment where resources are scarce (i.e., the economy is in recession), we often see a boom in startups, as in post-200820. High unemployment and limited options led some to be more willing to take on risks such as starting their own business. In short, their opportunity cost has lowered. Similarly, with more academic researchers competing for shrinking federal funding resources, taking on the \"risk\" of investigating a rare disease indication, which traditionally would have been unappealing due to the lack of resources or a clear career path, has become more desirable. As many trained scientists leave academia, this may be an opportunity to draw their attention and expertise to rare diseases through resources like patient advocacy groups which in turn make some funding available. The decrease in traditional opportunities can then be coupled with the lower cost of getting started in rare disease research. For example, because less research has been done in these orphan areas, some of the most basic (and less expensive) research has yet to be performed. Initial genomic sequencing and analysis for mutations, for example, is cheaper and faster than the more advanced work that is the initial starting point in other more common diseases. Rare disease advocates often must be more imaginative and innovative in order to leverage their limited resources, and in this case, brain power (in the form of brilliant researchers) is a resource that the rare disease community can leverage by presenting a strong case for the career and research opportunities available by focusing in an orphan area.\n\nAn additional approach for small rare disease foundations is to pool their resources, and that could be at the level of organizational staff for fund raising or at the scientific level. For example, one experienced scientific consultant could oversee the science collaborations for multiple distinct organizations dealing with the same or different rare diseases. This not only has cost savings but also the potential to see synergies across projects and research. This may increase the potential for serendipitous discovery that might synergize the overall research goals for multiple diseases. Obviously, there need to be boundaries to respect the intellectual property of groups involved, but the benefits may outweigh the risks.\n\n\nA coordinated research effort\n\nFrom our own experience of working as researchers or facilitators of collaborations between different groups working on rare diseases, we will now describe some of the results of ongoing efforts. The aim is to give the reader some understanding of the breadth of technologies applied and the number of approaches being worked on simultaneously. Recent examples suggest that through collaborative efforts, more progress can be made.\n\nSpinal Muscular Atrophy (SMA) is a childhood-onset, neurodegenerative disorder that is characterized by the loss of motor neurons and affects approximately one in 11,000 people. The disease has a range of clinical presentations, which are categorized into four types21,22. Type I, the most severe form that represents approximately 60% of cases, is diagnosed prior to six months of age, and patients do not gain the ability to sit. In 1995, researchers uncovered the genetic basis of the disorder, which accounts for at least 95% of cases, mutations in the Survival of Motor Neuron1 (SMN1) gene23. In addition to SMN1, humans have a variable number of copies of the SMN2 gene, which differs from SMN1 by a single nucleotide, and leads to a change in the splicing pattern, resulting in a truncated form of the protein that is quickly degraded24,25. The severity of SMA is determined by the number of copies of SMN2, with type I patients having fewer copies. This knowledge allowed for the development of animal models26–28 and drug discovery assays based on splicing modification and the levels of SMN29. In addition, basic science understanding such as the function of the SMN protein could be probed. Answers to important questions for translational decisions, like where in the body SMN levels must be increased (both in neurons and peripherally) and when treatment is required for response (early intervention is better, but rescue is possible after onset of symptoms) have also been addressed30,31. Despite the complexity of the SMN protein and the disease pathology, with a clear directive in mind such as to increase levels of SMN by inducing transcription, changing splicing, or preventing protein degradation, the research community has many interesting findings to date which have resulted in five promising drug candidates in clinical trials and at least 11 preclinical programs29. Four out of the five clinical candidates are directly targeting SMN expression, either through gene therapy or modulation of SMN2 transcription or splicing with small molecules or an antisense oligonucleotide (ASO)30,32–37 (Table 2). The furthest along of these candidates is the ISIS-SMNRx ASO, being developed by ISIS Pharmaceuticals and Biogen-IDEC, which is currently in a Phase III trial38.\n\nSMA research in academia and in industry has been strongly supported and guided by the SMA Foundation39 and CureSMA40 (formerly known as Families of SMA Foundation), which both coordinated research efforts, fostered collaborations, enticed biopharma companies, and developed an extensive patient network for clinical trials. Through these foundations, patient involvement in research was critical for genomic studies, understanding the natural history of SMA, development of induced pluripotent stem cells for disease modeling, clinical trials, and identifying biomarkers. Collaborative partnerships between academia, government, pharmaceutical companies, and non-profits accelerated efforts in compound screening on biochemical and cellular assays, animal testing, and other aspects of drug development, have led to the creation of a robust pipeline over a fifteen year period.\n\nCMT affects approximately one in 2500 Americans41. Patients usually have muscle weakness, which results in difficulty walking and gripping objects and progresses to foot and hand deformities, decreased reflexes, and bilateral foot drop42. In most cases, the cause is genetic but it can also be induced by other factors such as certain chemotherapy drugs. The Peripheral Myelin Protein 22 (PMP22) gene duplication predominantly causes the most common form of CMT called CMT1A4,43. There is no treatment for any of the CMTs for which symptoms usually present in the first two decades of life44.\n\nDespite discovery of the causal gene duplication in 1991, the first high throughput screen (HTS) targeting PMP22 was not published until 21 years later in 201245. Thus, CMT1A is one of many rare disorders where fundamental discoveries in academia progress slowly towards the therapeutic development. Still, the pipeline for CMT1A looks quite promising with Pharnext announcing a phase III clinical trial for PXT-3003 (a combination therapy of FDA-approved components baclofen, naltrexone and sorbitol) in 2014–201546,47. If the Phase III clinical trial is ultimately successful, this could be the first treatment to market. Still, success is not a given, as success rates for investigational drugs in phase III trials from a recent analysis was 60.1%48. The pharmaceutical company Addex announced a preclinical study of ADX71441, a GABA-B receptor (GABA-BR) positive allosteric modulator (PAM) compound49. Most recently, researchers at the Max Planck Institute (Germany) announced how Neuregulin-1 might represent a promising approach to therapy50. These latter two therapies are likely many years from the clinic.\n\nThere are two foundations focused on developing treatments for CMT. The first is the Hereditary Neuropathy Foundation (HNF)51. This group has funded a number of model systems to further research, including the development of a high content screen of over 25,000 compounds for CMT1A (PMP22) and CMT1E (point mutation), the establishment of transgenic CMT1A rat models, a CMT2A (MFN2 mutation) mouse model for testing therapeutics, and a CMT2A zebrafish model for screening. HNF also developed a global registry for inherited neuropathies19. Recently HNF partnered with Pharnext to raise awareness of CMT1A in preparation for their phase III clinical trial. The Charcot-Marie-Tooth Association52 has also raised funds to develop laboratory models and assays at academic partners, perform HTS screening at the National Institutes of Health (NIH) and with pharmaceutical companies (it recently announced a partnerships with Genzyme and Addex). To date the most advanced work has focused on PMP22 for CMT1A. The CMTA has also set up a relationship with a contract research organization to perform drug testing in laboratory models of CMT1A. This foundation also funds work on other CMT forms but this appears to be at an earlier stage than for CMT1A.\n\nThe Inherited Neuropathy Consortium Rare Diseases Clinical Research Network (RDCRN) is an NIH collaboration between CMT researchers. Over the past five years, with funding likely in excess of $5 million, this network has focused on determining the natural history of CMT through clinical (https://www.rarediseasesnetwork.org/INC/studies/index.htm) projects and may be engaged in the future for testing treatments.\n\nAt first glance this represents an incredible amount of activity for CMT, but it is worth also considering that there have been considerable failures such as the use of high-dose ascorbic acid for CMT1A53,54. The heavy focus on PMP22 is a risk given the heterogeneity of the disorder, and this could be mitigated in some way by more collaboration between researchers and foundations to avoid potential for overlap and also explore new approaches. Screening more compounds against PMP22 is likely not going to lead to more insights, and learning from the data already generated via computational modeling would perhaps be beneficial. CMT research is not unique amongst rare diseases in having trouble translating discoveries in the lab into the clinic. It is clear that millions of dollars can be invested by both foundations and the NIH with no guarantee of a treatment resulting from it. Rare disease patients and foundations need to have realistic expectations of the length of time it takes to go from a HTS to the clinic.\n\nGiant Axonal Neuropathy (GAN) is a recessively inherited condition that results in progressive nerve death55, and it has been reviewed previously9,19. GAN may be closely related to Charcot-Marie-Tooth Type 2 (CMT Type 2)56, and some pathological factors are also hallmarks of amyotrophic lateral sclerosis (ALS or Lou Gehrig’s Disease)57, CMT 2E58, Alzheimer’s disease, Parkinson’s disease, diabetic neuropathy, SMA, as well as other diseases59. A parent/patient led foundation, Hannah’s Hope Fund (HHF)60 has raised over $5 million to fund the development of a gene therapy61. In addition, they are also funding a postdoc at National Center for Advancing Translational Sciences (NCATS) to develop an assay and screen compounds. This illustrates what can be achieved in a short period of time by promoting collaboration between different academic and government research groups9,19 and perhaps represents a model that other groups could emulate.\n\nSanfilippo syndrome (mucopolysaccharidosis type III; MPS III) is a devastating neurodegenerative lysosomal storage disorder of childhood. The cause of MPS III is an inherited mutation in one of four enzymes required to catabolize heparan sulfate (HS). The four subtypes of the disease are defined by the enzyme deficiency: MPS III type A (heparan N-sulfatase); MPS III type B (α-N-acetylglucosaminidase); MPS III type C (heparan sulfate acetyl CoA: α-glucosaminide N-acetyltransferase, HGSNAT); and MPS III type D (N-acetylglucosamine 6-sulfatase)62. All subtypes of MPS III have similar clinical phenotypes with onset in infancy or early childhood: progressive and severe neurological deterioration, hearing loss, and visceral manifestations62. There is currently no cure or effective treatment available for MPS III. There are however many therapies in early development (Table 3), including gene therapies, enzyme replacement, chaperone and substrate reduction therapeutics. With Sanfilippo MPSIII Type A there is currently a large focus on gene therapy evaluating different vectors (adeno-associated virus (AAV) e.g. AAV5, AAV9, AAVrh.10 etc) across many different groups. Less research appears to be focused on types C, D (Table 3). Due to the limited pool of funding for this disorder, enhanced collaboration may prevent unnecessary redundancies and broaden the impact of the ongoing research efforts as well as make the investments go further.\n\nThis is by no means exhaustive and we are aware of other efforts, but these are not public knowledge in many cases. (This table is an updated version of that found at https://www.rareconnect.org/en/community/sanfilippo-syndrome/article/current-sanfilippo-research-programs-in-the-clinical-stage).\n\nThe primary goal of rare disease research is to find a cure or treatment strategy for the disorder in question, but rare diseases offer a glimpse into the roles of genes and proteins in human disease pathogenesis in general. The therapeutics developed to treat rare disorders may also be useful in treating additional disorders whether rare or common. This has proven to be true for drugs developed for rare cancer indications. For example, imatinib (Gleevec®) was originally approved for Philadelphia chromosome positive CML, which contains the oncogenic BCR-ABL tyrosine kinase mutation63,64. Imatinib is a well-absorbed drug with activity against multiple tyrosine kinases beyond BCR-ABL, including c-KIT and PDGFRA65,66. Due to these other activities, imatinib is effective at treating gastrointestinal stromal tumors (GIST) which are dependent on c-KIT, many other cancers, and steroid refractory Graft-versus-Host disease which requires PDGFRA activity67. Thus drugs developed for one rare disease can serve broader roles based on related biological mechanisms.\n\n\nSoftware for collaborations\n\nThere appears to be increased interest in scientific collaborations on a large scale and developing a software to facilitate this68. For rare diseases, collaborations even on a small scale could have real impact. Instead of scientists hoarding their data, we could remove unnecessary duplication and speed development.\n\nResearch collaborations are seen as important for drug discovery to speed up biomedical research, reduce costs, and prevent unnecessary repetition of experiments69. There are however considerable intellectual property (IP) concerns to be overcome when sharing data70,71. Increasingly, pharmaceutical companies are involved in multi-organization collaborations and public-private partnerships (PPP). To address these issues, Collaborative Drug Discovery, Inc. (CDD) created a software which enables researchers to have their own private vault for storing chemistry and biology data, which can be securely shared and mined while maintaining IP status72. CDD itself has found a niche in hosting large-scale collaborations such as More Medicines for Tuberculosis (MM4TB), Bill and Melinda Gates Foundation (BMGF) TB Accelerator73, and the NIH Blueprint for Neuroscience Research (BPN). In addition, rare disease research organizations, such as the Myelin Repair Foundation (MPF) and Jonah’s Just Begun, have used CDD to manage ongoing drug discovery efforts. CDD has a trove of public information, which provides datasets that can be useful for rare disease researchers. These include FDA approved drugs and compounds that have been identified by in vitro screening for repurposing74, and the National Center for Advancing Translational Sciences (NCATS) molecules for repurposing75. CDD has recently added the NIH's Molecular Libraries Probe Production Centers Network (MLPCN) probe compounds alongside the scoring of these molecules by an experienced medicinal chemist76. Comparison of these public datasets with private data may lead to novel drug repositioning ideas, which may in turn mean an accelerated path towards new treatments74,77,78. Many academic screening centers are focused on repurposing current FDA approved compounds79, so the missing piece is developing phenotypic or target based screens for more rare diseases.\n\nBeyond the desktop, we must seriously consider how mobile devices could be used to share data and foster collaborations in rare diseases. A mobile app called Open Drug Discovery Teams (ODDT)80,81 was created to collect Twitter feeds on multiple scientific hashtags (e.g., rare diseases like #huntingtons, and #sanfilipposyndrome, #gaucher, #huntersyndrome #fabry, #Tay-Sachs, #NGLY1, #hurlersyndrome, #pompedisease, #krabbe, #fmdaware, #niemannpick and #Batten). Collecting tweets and information from the web creates an open database for these diseases. The architecture of ODDT has been described recently82, and the use and function of the app have also been discussed80. The app is also small molecule aware so it can be used to share structures and activity data. The limited funding available for rare disease drug discovery and development suggests why we should be looking at alternative, lower cost approaches. ODDT could even become a useful assistant to scientists, small rare disease foundations, and advocates to help find collaborators or groups to fund. ODDT is ultimately a simple tool that uses Twitter for serious science applications that could be expanded in several directions to help the rare disease community. For example, besides collaboration towards one rare disease treatment, there is also opportunity for rare disease researchers to work together to compare HTS drug discovery data. Can rare disease groups learn from one another? Could hits found for related rare diseases have additional applications, or might safety and toxicity issues be determined earlier if the data were compared sooner?\n\n\nWhat is still needed\n\nHow can treatment options be identified and/or created in a patient relevant time frame? The most obvious way is to identify an approved medicine that could be repurposed, or if warranted, used off label. Another option is to discover a medicine specific for that disease. Currently only few medicines are discovered each year for rare diseases. A recent analysis showed only 46 first in class medicines approved for rare diseases in a 14 year period3. With almost 7000 rare diseases, it is impossible to discover medicines for all of these using current research practices. Clearly, the number of identified human rare disease genes significantly outstrips the number of global research laboratories available to investigate a given disorder. The current productivity of drug discovery will never fill this need. Therefore, the development of a strategic toolbox and preclinical research pathway for inherited rare diseases has been proposed83.\n\nThe unfortunate reality of drug discovery as it is currently practiced is it is a long and costly process. Increased success in rare disease drug discovery will require better diagnostics, an understanding of disease that provides good translational biomarkers, and clearer clinical development programs. The mechanisms underlying rare diseases are not well understood, patients are hard to identify and diagnose, and no regulatory precedent for the disease may exist, all of which makes designing and conducting drug development programs very difficult. There has been a dramatic increase in research and development spending without the corresponding increases in new medicines. The current trend is to spend more to increase knowledge, however this has not increased the clinical success rate. The low productivity is unacceptable for rare disease drug discovery. Funds need to be used more efficiently. The new knowledge needs to be used more effectively to identify treatment options. Solutions that provide for more treatment options in addition to new medicines are needed. Some hope in rare diseases is provided by understanding the genetic cause to the disease. As noted above, greater than 80% of rare diseases are due to a genetic defect. This understanding can focus research efforts and inform potential medical treatments. However, knowing the identity of a casual gene does not readily lead to a medicine that will cure the disease. Opportunities exist to use the genetic information to provide treatment options, in addition to informing drug discovery research.\n\nWhat are the options if there is not the time or funding available to discover and develop new medicines? One potential option is to identify molecules approved for human use including pharmaceuticals, nutraceuticals and herbal products that can be safely given to patients. While this seems obvious, there is a huge barrier to the dissemination of information between researchers and patients, physicians and advocates to develop a treatment plan based on all the available information. For rare disease researchers, a comprehensive data management system that consolidated the underlying genetic and protein causes of these disparate rare diseases would be hugely useful. Bringing these data together in a comprehensive database with information that reaches beyond just the underlying gene to other biological relevant information such as pathway analysis will be critical to researchers performing drug discovery on these disparate diseases. In parallel, the patient of a child with a rare or ultra-rare disease has few options in the USA as there is currently no single entity that covers all rare disease research and clinical translational work. Ideally, a comprehensive database would be presented to patients and advocates in a factual manner that is easy to understand, difficult to misinterpret, and could lead to connections with scientific and medical experts.\n\nThe assimilation and dissemination of knowledge from the many scientific areas important to medicine, including genetics, biology, chemistry and pharmacology, is challenging even for experts. Tools that provide this information to patients, physicians and advocates will be of value to help provide insights into new treatment options and to identify new opportunities. For example, providing information on approved medicines or remedies in which the pharmacology could be related to a specific physiological system and/or gene may identify new treatment options and/or new research directions. One approach to address this need is with an in silico database, in which the knowledge is easily used by both professionals and non-professionals. We envision that the identification of a new gene and the corresponding biology may provide insights into pharmacology that may be addressed with approved medicines. This knowledge could be of use to identify compounds for testing in animal and cellular studies. There is also the possibility of off label use in the patients with the proper monitoring, if no other treatment options are available.\n\nIt can be a challenge to match pharmacology with biology and genetics, especially for non-experts. Even domain experts in genetics and biology, may not know the corresponding pharmacology and vice versa. A database or collaborative network that specifically provides this information and access to experts will be of value to patients with rare diseases. For example, commercially available databases already exist that relate a gene to biological pathways with known pharmacology84 and could help identify treatment options amongst FDA-approved drugs, nutraceuticals or other compounds.\n\nThe details of hereditary angioedema (HAE) provide a nice example of how the identification of the mutated genes led investigators to identify the biological systems involved, which in turn provided clues to potential pharmacological intervention and approved therapeutics85. HAE is a rare genetic disorder that leads to episodes of extreme swelling caused by mutations to C1-esterase-inhibitor (C1-INH), a protease inhibitor that functions in the complement cascade in the immune system86. HAE is characterized by low levels of C1-INH activity and low levels of C4 in the classical complement pathway87. C1-INH regulates the activation of complement and intrinsic coagulation (contact system pathway), and is a major endogenous inhibitor of plasma kallikrein. The kallikrein-kinin system is a complex proteolytic cascade involved in the initiation of both the inflammatory and coagulation pathways. One critical aspect of this is the conversion of High Molecular Weight (HMW) kininogen to bradykinin by the protease plasma kallikrein. In HAE, normal regulation of plasma kallikrein activity and the classical complement cascade is not present. During attacks, unregulated activity of plasma kallikrein results in excessive bradykinin generation. Bradykinin is a vasodilator which is thought to be responsible for the characteristic HAE symptoms of localized swelling, inflammation, and pain88. Two treatments for acute episodic attacks of HAE were developed once the causative gene was uncovered and required an in-depth understanding of the biology. Ecallantide binds to plasma kallikrein inhibiting the conversion of HMW kininogen to bradykinin89. Icatibant is a competitive antagonist selective for the bradykinin B2 receptor90–92. Thus, this provides a clear example of how genetics can be connected to known pharmacology.\n\nSt. Jude Children’s Research Hospital, which from humble beginnings has transformed many cancers of children into treatable diseases through combined basic and translational research while at the same time becoming a world class center of excellence, offers a glimmering example or model for the rare disease community. Perhaps a dedicated rare disease institute to help facilitate and organize collaboration for translational research could be valuable. The development of such a center would need coordination between foundations, philanthropists, researchers, and government to ensure that it could become a reality.\n\nIt is our opinion that we need to centralize many of the rare disease efforts and translate findings to other rare diseases where there may not be current organizations driving the research. These efforts could include the development of databases of transcriptional profiles for thousands of compounds which many pharmaceutical companies have access to. Computational advances could be used in so many areas that would help rare disease research. This might include improving the prediction of small molecule-RNA/protein interactions, generalizing ADME-toxicology for oligonucleotides, or possibly identifying a druggable pathway that allows the persistence of higher levels of mutated and mis-folded protein. These may be just starting points for additional investments. With so many in silico cheminformatic and bioinformatic methods93,94, bringing them together via data integration platforms like those for systems biology, could help areas of research such as chaperonin identification74,95. Unfortunately, there is currently no definitive database for collaboration and education that disseminates available knowledge to rare disease stake holders (patients, physicians, advocates) in a usable/interpretable form. Such a database may provide insights into additional treatment options in a time frame relevant to patients.\n\nAn institute for rare diseases, which could be informatics driven, to centralize and direct the various ongoing academic collaborations funded by the rare disease groups would be a huge advance for optimal collaboration. Such an effort could use the various existing databases to mine for compounds as potential treatments for rare diseases. A recent effort to collate 456 FDA compounds approved for use in the pediatric population may be a starting point for repurposing these compounds for rare diseases computationally96. An institute would partner with a center with HTS screening resources and would leverage existing infrastructure and researchers across many other institutes. Rare disease patient groups would be targeted to provide foundational funding and access to their complete researcher and patient networks. Pharmaceutical companies would be involved to provide access to compounds and databases for mining. In addition, advice from a scientific advisory board of experienced drug developers would be critical. The institute would share IP with the groups involved. The goal would be the creation of a world class center for rare diseases, becoming a magnet for global rare disease researchers, clinicians, patients, and companies, and it would be self-funding. The ultimate measure of its success would be how many treatments for rare diseases would be approved.\n\n\nA hopeful future\n\nThe features of rare diseases that lead to their unique challenges can also become advantages for finding new therapeutics. Once united, a well-defined patient population, a defined genetic etiology, and a dedicated advocacy foundation can catalyze drug discovery. Collaboration between all of the key entities (Figure 1), including academic institutions, government, biopharmaceutical companies, advocacy organizations, and non-profits is critical for moving rare disease drug discovery efforts forward. In addition, computational approaches can help foster the collaborations, add efficiency, build on previous efforts, and ultimately drive research in new directions. Individual rare disease researchers may also benefit from working together, perhaps through a centralized institute, to share resources towards the ambitious goal of finding treatments for the large unmet need.", "appendix": "Author contributions\n\n\n\nN.K.L. and S.E. came up with the general idea for the Opinion Article. All authors contributed to the collaborative writing of this project.\n\n\nCompeting interests\n\n\n\nS.E. works for Collaborations in Chemistry, and consults for Collaborative Drug Discovery Inc. as well as rare disease groups including the Hereditary Neuropathy Foundation, Hannah's Hope Fund and Phoenix Nest. N.K.L. works for Collaborative Drug Discovery Inc. M.R. works for National Brain Tumor Society. D.C.S. works for the Institute for Rare & Neglected Diseases Drug Discovery.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nThe authors would like to acknowledge Jill Wood and Raquel Marques (Jonah's Just Begun-Foundation to Cure Sanfilippo Inc.) for assistance compiling Table 3. Lori Sames (Hannah's Hope Fund) and Allison Moore (Hereditary Neuropathy Foundation) are also acknowledged for their support and information that enabled the summaries of GAN and CMT, respectively. Our colleagues at CDD are acknowledged for their efforts in developing the CDD Vault. Dr. Alex Clark is kindly acknowledged for co-developing the ODDT mobile app.\n\n\nReferences\n\nMelnikova I: Rare diseases and orphan drugs. Nat Rev Drug Discov. 2012; 11(4): 267–8. PubMed Abstract \| Publisher Full Text\n\nField MJ, Boat TF: Rare Diseases and Orphan Products: Accelerating Research and Development. 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Publisher Full Text\n\nEkins S, Hohman M, Bunin BA: Pioneering use of the cloud for development of the collaborative drug discovery (cdd) database in Collaborative Computational Technologies for Biomedical Research. S. Ekins, M.A.Z. Hupcey, and A.J. Williams, Editors. Wiley and Sons: Hoboken. 2011.\n\nBunin BA, Ekins S: Alternative business models for drug discovery. Drug Disc Today. 2011; 16(15–16): 643–645. PubMed Abstract \| Publisher Full Text\n\nHohman M, Gregory K, Chibale K, et al.: Novel web-based tools combining chemistry informatics, biology and social networks for drug discovery. Drug Disc Today. 2009; 14(5–6): 261–270. PubMed Abstract \| Publisher Full Text\n\nEkins S, Bunin BA: The Collaborative Drug Discovery (CDD) database. Methods Mol Biol. 2013; 993: 139–54. PubMed Abstract \| Publisher Full Text\n\nEkins S, Williams AJ, Krasowski MD, et al.: In silico repositioning of approved drugs for rare and neglected diseases. Drug Disc Today. 2011; 16(7–8): 298–310. 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Reference Source" }	[ { "id": "6611", "date": "18 Nov 2014", "name": "Nicholas Meanwell", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well-written and articulate article advocating collaborative research and organizational approaches to develop drugs with the potential to treat rare diseases. The case for collaboration is thoughtfully developed and the authors make compelling arguments. I fully sympathize with the contentions laid out in the article and this piece will be a valuable addition to the literature.", "responses": [ { "c_id": "1248", "date": "03 Mar 2015", "name": "Sean Ekins", "role": "Author Response", "response": "Thank you for your review and support of this article, it is greatly appreciated." } ] }, { "id": "7513", "date": "28 Jan 2015", "name": "Stephen Groft", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very well referenced opinion article on rare diseases and orphan products including historical and current activities that have led to an increased global emphasis on rare diseases research and orphan products development. The article highlights several of the extremely important aspect of research and development for products for rare diseases – collaboration and coordination of activities. Often, the coordination is led by the leadership of the patient advocacy groups.\n\nSince very few research locations have sufficient patient populations to initiate patient recruitment sufficient to open and complete the study, a critical mass of investigators must be established from USA and international research sites. Likewise, patients and patient advocacy groups are now recognized key partners in the research continuum facilitating the recruitment of patients and providing active liaison services with the academic research community, biopharmaceutical industry, media, Government research and regulatory agencies, and the public.The article highlights several rare diseases requiring coordinated activities leading to research advances for several rare diseases, e.g., Spinal Muscular Atrophy, Charcot-Marie-Tooth disease, Giant Axonal Neuropathy, Sanfilippo syndrome. These disorders have unique requirements in research design and initiation of clinical trials with appropriate plans for data collection and analyses. The requirements necessitate early and constant contact with the regulatory agencies to reach agreement on biomarkers and clinical or surrogate endpoints for the study, the number of patients required for the study or clinical trial, and the analyses of data to be used to establish the safety and efficacy of the product prior to patient and physician access for patient care. This emphasis on expanded interactions with regulatory agencies has been presented in the article and is particularly important for consideration by the reader as FDA has added many unique regulatory approaches to product review, such as, priority and expedited review and approval of products, fast track review, and breakthrough therapy designation. It is important to understand the value of these regulatory programs and benefits accompanying these products in the research and review stages of product development.The authors’ opinion article indicates one of the great barriers to the development of orphan products has been the financial risk of developing a product for a disease with very small patient populations of potential users. The collaborative method of research and development will spread the financial risk over many partners especially in the pre-clinical and early clinical stages of development. For some rare diseases, industry partnerships have been easier to obtain after certain milestones have been exceeded and the likelihood of clinical success is apparent with limited or relative toxicity expected. Reaching these milestones have been reported as significant in the decision making process. For most rare diseases, sharing of global resources remains a powerful incentive for biopharmaceutical industry participation.The authors conclude with suggested future activities including the dissemination of information to patients and clinicians about novel treatments of rare diseases with selected sub-population of patients with specific genetic mutations. Also, the authors suggest a continued and expanded centralization of many of the rare disease activities including the development of an informatics driven initiative. The authors provide a hopeful view of the future based on even greater global coordination and collaboration offered by centralized sources of information and readily available shared resources from multiple resources in the public and private sectors.", "responses": [ { "c_id": "1247", "date": "03 Mar 2015", "name": "Sean Ekins", "role": "Author Response", "response": "We would like to thank you for taking the time to review and provide positive comments. It is very much appreciated!" } ] } ]	1	https://f1000research.com/articles/3-261
https://f1000research.com/articles/3-208/v1	02 Sep 14	{ "type": "Research Article", "title": "Stress, rejection, and hormones: Cortisol and progesterone reactivity to laboratory speech and rejection tasks in women and men", "authors": [ "Allison E. Gaffey", "Michelle M. Wirth", "Allison E. Gaffey" ], "abstract": "Stress and social rejection have important impacts on health. Among the mechanisms implicated are hormonal systems such as the hypothalamic-pituitary-adrenal (HPA) axis, which produces cortisol in humans. Current research employs speech stressors and social rejection stressors to understand hormonal responses in a laboratory setting. However, it is not clear whether social rejection stressors elicit hormonal reactivity. In addition to cortisol, progesterone has been highlighted as a potential stress- and affiliation-related hormone in humans. In the present study, 131 participants (70 men and 61 women) were randomly assigned to be exposed to one of four conditions: standardized speech stressor; speech control; social rejection task; or a control (inclusion) version of the social rejection task. Saliva samples were collected throughout the study to measure cortisol and progesterone. As hypothesized, we found the expected increase in cortisol in the speech stressor, and we also found that the social rejection task did not increase cortisol, underscoring the divergence between unpleasant experiences and HPA axis activity. However, we did not find evidence for progesterone increase either during the speech- or social rejection tasks. Compared with past studies on progesterone and stress in humans, the present findings present a mixed picture. Future work is needed to delineate the contexts and types of manipulations which lead to progesterone increases in humans.", "keywords": [ "human", "cortisol", "progesterone", "stress", "hypothalamic-pituitary-adrenal axis", "social rejection", "Cyberball", "Trier Social Stress Test" ], "content": "Introduction\n\nThere is a growing interest in human behavioral endocrinology. Encouraged by the availability of non-invasive salivary hormone measurements, researchers in clinical, social, and personality psychology, among other fields, are increasingly incorporating hormonal measurements into their research in order to discover the impact of stress and other kinds of social or emotional stimuli on hormonal systems in human beings.\n\nAmong many hormone-relevant psychological constructs, affiliation and bonding, and the converse, isolation and rejection, have received particular attention. Loneliness, lack of social support, and ostracism are known to have grave psychological and health impacts over time (see e.g. Hawkley & Cacioppo, 2010 for a review). Activation of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in high levels of glucocorticoids, has been proposed as one possible mechanism mediating the connection between isolation and poor health (Hawkley & Cacioppo, 2010). This idea is supported by evidence that loneliness correlates with higher levels of cortisol, the primary glucocorticoid in humans (Hawkley & Cacioppo, 2010); the fact that isolation is a potent stressor and elicitor of glucocorticoid release in other social animals, such as rats and sheep (e.g., Hermes et al., 2009; Rivalland et al., 2007); and that chronically high glucocorticoid levels are linked to a number of health consequences (Sapolsky, 2002; Tsigos & Chrousos, 2002). However, the relationships between isolation/rejection, HPA axis activation, and health are still not understood completely. It is necessary to study these relationships on both a macro-level in real-world, longitudinal data and also at a micro-level in controlled laboratory settings in order to precisely define the mechanisms involved.\n\nResearchers have used laboratory rejection tasks such as Cyberball — a ball-playing game in which other players exclude the participant — in order to test both the psychological and hormonal effects of rejection (Maner et al., 2010; Stroud et al., 2002; Williams et al., 2000; Zwolinski, 2012). However, studies have failed to find consistent hormone responses to rejection (Zwolinski, 2012). There has also been evidence of sex differences in hormonal responses to rejection (Stroud et al., 2002), but these effects were not replicated in a separate study (Linnen et al., 2012). It is important to determine whether rejection in a laboratory setting can elicit an HPA axis response, and if so, in which sex or sexes.\n\nPsychological factors known to influence the HPA axis include novelty, unpredictability, and a lack of control (Mason, 1975). A more recent meta-analysis identified social-evaluative threat as key in predicting HPA axis responsivity to laboratory stress tasks (Dickerson & Kemeny, 2004). Any or all of these factors might be present to some degree in a rejection situation, so a cortisol response to rejection in the laboratory could be expected. On the other hand, the main function of glucocorticoids is to mobilize energy, e.g. for fight-or-flight activities (Nelson, 2005; Sapolsky, 2002; Wirth & Gaffey, 2013). Therefore, glucocorticoids do not show a one-to-one relationship with negative affect, but instead are elevated in situations requiring energy, whether associated with negative affect or not; some examples include sickness, exercise, and giving a speech (Wirth et al., 2011; Wirth & Gaffey, 2013). Whereas commonly-used speech stressors require literally thinking on one’s feet and making a vigorous (and ultimately futile) attempt to impress the judges, social rejection in laboratory tasks like Cyberball may or may not demand any expenditure of energy – in fact, it may be a situation in which no obvious actions can be taken. Therefore, it is unclear whether laboratory social rejection is a context in which the brain and body would activate a system designed to replenish energy. The first goal of the present study, then, is to examine the effect of a popular rejection manipulation, Cyberball (Williams et al., 2000), on cortisol levels in men and women, alongside the effect of a well-studied, standardized laboratory stressor, the Trier Social Stress Test (TSST; Kirschbaum et al., 1993).\n\nIn addition to cortisol, there is a growing body of literature linking progesterone levels/responses to both stress and to affiliation and rejection (Brown et al., 2009; Childs et al., 2010; Gettler et al., 2013; Maner et al., 2010; Schultheiss et al., 2003; Schultheiss et al., 2004; Wirth & Schultheiss, 2006; Wirth et al., 2007; Wirth, 2011). Progesterone is not only a gonadal hormone, but is also produced in the adrenal glands, and progesterone levels increase in response to pharmacological stimulation of the HPA axis (Genazzani et al., 1998). Progesterone and hormones synthesized from it (e.g., allopregnanolone) increase during stress in laboratory animals (Barbaccia et al., 2001; Paul & Purdy, 1992; Purdy et al., 1991), but it is as of yet unclear whether progesterone is part of the typical human stress response (Wirth, 2011). There is evidence that progesterone does increase alongside cortisol during venipuncture stress (Wirth, 2011), and also evidence that progesterone responds to the TSST stressor, at least in men, and in women in some menstrual cycle phases (Childs et al., 2010). Progesterone responses to laboratory stressors need to be studied systematically in both sexes, in part simply to understand stress physiology, but also because of important implications for understanding psychological disorders (see Wirth, 2011 for a review). Furthermore, progesterone might be particularly associated with affiliation and rejection/isolation, as detailed below.\n\nAlthough cortisol and progesterone levels seem to rise and fall in tandem in humans (Wirth et al., 2007), a growing body of literature supports associations with affiliation that are unique to progesterone. First, implicit affiliation motivation – a personality construct measuring drive for friendly, warm contact with others - was increased in women taking oral contraceptives containing progestins, as well as in cycling women in the luteal phase, a time in the cycle of high progesterone levels (Schultheiss et al., 2003). Second, a rejection-themed film excerpt designed to produce affiliation-related stress caused increases in progesterone as well as cortisol; in addition, baseline (pre-film) affiliation motivation predicted stress-related increases in progesterone (but not cortisol), without regard to participant sex (Schultheiss et al., 2004; Wirth & Schultheiss, 2006). Third, women who took part in a closeness-generating task in pairs had progesterone increases in response to the task, compared to a control condition (Brown et al., 2009). Fourth, personality traits such as social anxiety and rejection sensitivity moderated progesterone responses to a laboratory rejection task (Maner et al., 2010). Finally, recent, preliminary research links progesterone to the beneficial effects of helping behavior on cardiovascular recovery from stress (Brown & Brown, 2011; Smith, 2011) and to positive mood during fathers’ interactions with their toddlers (Gettler et al., 2013).\n\nGiven this evidence, along with evidence that progesterone may respond to typical laboratory stressors (Childs et al., 2010; Wirth, 2011), is not yet clear whether progesterone is a “generic” stress hormone, i.e. responding to all stressors along with cortisol, or whether it is tied specifically to affiliation stress/rejection. Notably, in some of the studies cited above, progesterone and not cortisol showed associations with affiliation (e.g. Wirth & Schultheiss, 2006). Thus, this evidence calls for further research elucidating progesterone’s role in stress, affiliation, and rejection. While there is at least one study of progesterone in the context of laboratory rejection tasks (Maner et al., 2010), moderating variables were the focus of that study; more work is needed to determine whether progesterone typically increases during rejection in human beings. Thus, the second goal of the current research is to test whether progesterone increases in response to either the rejection manipulation Cyberball, and/or a standard speech stressor (the TSST).\n\nIn both goals of the present research, it is important to determine if there are sex differences. Men typically have larger cortisol responses to laboratory stressors than women do, despite women having equivalent, or even greater, self-reported mood responses (Kudielka & Kirschbaum, 2005). On the other hand, women are thought to be more sensitive to rejection than men (Stroud et al., 2002). In addition to cortisol, progesterone responsivity to both rejection and a speech stressor may have important sex differences (e.g., Childs et al., 2010). For these reasons, we collected data in both women and men exposed to Cyberball or the TSST.\n\nOur hypotheses were four-fold. We expected to (1) replicate substantial prior research (Dickerson & Kemeny, 2004; Kirschbaum et al., 1993; Kudielka & Kirschbaum, 2005) in that the TSST would cause increases in cortisol, particularly in men. We further hypothesized (2) that the TSST would have a greater effect on cortisol than would Cyberball, as the latter is not associated with clear needs for energy mobilization. As for progesterone, we hypothesized that (3) it would increase alongside cortisol in the TSST, as seen in men in at least one previous study (Childs et al., 2010). Given evidence for particular associations with rejection, we also hypothesized that (4) progesterone levels would be affected by Cyberball. We were agnostic as to whether this effect would be present in both sexes, given the paucity of published data on this topic.\n\n\nMethods\n\nUndergraduate students (N = 142: 71 men: Mage = 19.51, SDage = 1.39; 71 women: Mage = 19.81, SDage = 2.43) were recruited through the University of Notre Dame Psychology Department study pool and through flyers advertising a paid research study open to nonsmoking individuals 18 and 35 years of age. Exclusion criteria included currently nursing or pregnant, and hormonal conditions such as thyroid disorders. In addition, 9 women were taking oral contraceptives and were excluded from analyses. Participants received study pool credit or a cash payment of U.S. $10/hour. The procedures were approved by the University of Notre Dame Institutional Review Board (Protocol #12-09-486), and all participants provided informed consent prior to participation. One man and one woman were excluded from all analyses due to minor changes in the protocol after their participation, leaving a final sample size of 131.\n\nData were collected between October 2010 and July 2011. Participants were asked to refrain from eating, drinking caffeine, brushing their teeth and vigorous exercise for 2 hours prior to the study. Participants completed one session, lasting 150 minutes, between 16:00 and 19:00 to minimize circadian ﬂuctuations in cortisol and progesterone (Dickerson & Kemeny, 2004; Groschl et al., 2003; Hansen et al., 2008; Nelson, 2005). Participants were randomly assigned to one of four conditions: 1) The “stress” condition of the Trier Social Stress Task, including an evaluated speech and difficult serial subtraction (TSST Stress; N = 36; Kirschbaum et al., 1993), 2) A “control” version of the TSST during which participants wrote an essay about their dream job and performed a simple addition task alone (without judges; TSST Control; N = 26), 3) The “inclusion” condition of Cyberball (Cyberball Control; N = 32) or 4) the “rejection” condition of Cyberball (Cyberball Rejection; N = 37) (Williams et al., 2000). To match the timing required for the TSST (15 minutes), prior to playing Cyberball, all participants wrote an essay about their dream job for 10 minutes; participants were informed that the essay’s content would not be judged or evaluated. The four tasks are further detailed below.\n\nUpon arrival to the laboratory, after obtaining written and verbal consent, participants provided a 5 mL saliva sample (~10 min. after arrival; see saliva collection methodology below) and completed initial questionnaires (~20 min. after arrival). Questionnaires assessed demographic information, affect, and factors that influence hormone levels such as sleep, exercise, and menstrual stage (see Supplementary file). A professional online survey distribution tool, the Qualtrics Survey Research Suite (Qualtrics, Provo, Utah), was used to capture all self-report data. After completing these initial questionnaires, participants provided a second saliva sample (~30 min.).\n\nParticipants were then given directions associated with their randomly assigned task (i.e. Cyberball or TSST) and condition (i.e., Stress/Rejection or Control) before providing a third saliva sample (~50 min.). All participants then engaged in one of the four task-condition combinations. After the Cyberball task, all Cyberball participants completed additional assessments of inclusionary status and ostracism used in previous research (Zadro et al., 2004). Example questions included evaluating the degree to which they “Felt like an outsider during the Cyberball game” and “To what extent did the other participants include you during the game?”\n\nFollowing the TSST task or Cyberball ostracism questionnaires, participants completed a fourth saliva sample (~70 min.). Participants provided their fifth saliva sample (~105 min.) and sixth and final saliva sample (~150 min.) interspersed among affect questionnaires and non-emotionally-arousing tasks used to test separate hypotheses. Finally, participants completed an open-ended question of any comments or notes about the study, as a suspicion check for Cyberball. The timeline of events in each study session is shown in Figure 1.\n\nS1, S2, etc. represent saliva samples; approximate times are shown for the study session on a 24-hour clock.\n\nTrier Social Stress Test (TSST). In the TSST (Kirschbaum et al., 1993), participants have 5 minutes to prepare a speech on a topic they are not well prepared for; in this study they were instructed to try to convince judges who were “experts in judging non-verbal behavior” that they were the best candidate for their dream job. Participants were instructed to only use true information about themselves in their speech. Just before giving their speech, participants’ notes were unexpectedly removed. Participants then gave their speech for 5 minutes in front of two judges trained to display flat affect (i.e. no smiling or nodding) and give prompts if the participant still had time remaining. Participants also were told they were being videotaped and were able to view themselves on closed-circuit computer monitor. Following the speech, participants completed a 5-minute difficult serial subtraction task out loud for the judges (e.g., count down from 1037 by 13’s). The judges required participants to start the task over whenever they made a subtraction mistake. Participants were fully debriefed at the end of the study that they had not, in fact, been videotaped, and that the judges were trained to display flat affect and otherwise increase the stress of the situation, rather than being experts in non-verbal behavior.\n\nMany different control conditions have been used for the TSST (see e.g. Kirschbaum et al., 1993; Het et al., 2009). In the present study, TSST Controls were asked to write an essay about their dream job. Experimenters informed participants in the TSST Control condition that the essay’s content would not be judged or evaluated. Additionally, TSST Control participants performed an easy counting task out loud (e.g., count down from 300 by 1’s) while alone in the TSST room. Thus, participants in this condition performed the same tasks as in the TSST Stress condition, but without pressure and without being watched or judged.\n\nCyberball. Cyberball is a computer “ball-toss” game during which participants are either included or ostracized by the other players in order to elicit feelings of social rejection (Williams et al., 2000). Participants in the Cyberball task were randomly assigned to either an inclusion (Control) condition, in which they were passed the ball equally often as the other players, or an exclusion/rejection condition, in which they were passed the ball equally often initially and then excluded from play for the rest of the game. Participants’ photographs were taken at the beginning of the session to accompany their character in the Cyberball game. Participants were told that the other two same-sex players (whose behavior was actually computer-generated) were located at another laboratory on campus. Before the game, experimenters made a fake phone call to the fictional lab; this call was intended to be overheard by participants to give the impression that the experimenters were synchronizing Cyberball players’ log-ins in the two labs. Names and photographs (students from another university) also accompanied computer players. As a supposed precaution, participants were asked to inform the experimenter at the beginning of the game if they knew the other participants. None of the participants indicated in their final study comments that they did not believe the Cyberball cover story. Participants were fully debriefed at the end of the study that the other players’ actions were generated by the computer. Participants played Cyberball for 5 minutes with two other players. The game was set for 100 throws.\n\nWe assessed cortisol and progesterone levels in the six saliva samples provided by each participant. Participants used passive drool into a straw (i.e., no gum, cotton, or other saliva flow stimulants) to deposit saliva into a test tube (typically, Ultra-High Performance 15 ml centrifuge tubes, VWR, Radnor, PA), and were allowed to drink sips of water following each sample. Tubes were capped and frozen at -18°C after each data collection session. After sample collection, saliva samples underwent three freeze-thaw cycles (i.e. samples were thawed until liquid and re-frozen until solid, twice) and centrifugation (10 min at 3000 rpm). Cortisol and progesterone levels were determined by solid-phase 125I radioimmunoassays (Coat-A-Count, Siemens Healthcare Diagnostics, Duluth, GA), using the protocol described by Wirth & Schultheiss (2006). Range of standards used was 0.5 to 50 ng/ml for cortisol and 5 to 400 pg/ml (i.e., 0.005 to 0.4 ng/ml) for progesterone. A total of 8 assays for each hormone were performed in order to assay all 852 samples. Mean intra-assay coefficients of variation (CV) across all 852 samples were 7.1% for cortisol and 19.9% for progesterone. (Since progesterone is present at much lower concentrations than cortisol, CVs are typically much higher than for cortisol; see e.g. [Wirth & Schultheiss, 2006]. Average CVs for progesterone in this range have been reported in the literature previously and have been associated with theoretically-supported positive findings [Brown et al., 2009]). Inter-assay CVs for Stress and Control combined pools of saliva averaged 5.3% and 1.9% for cortisol, and 8.4% and 10.1% for progesterone. Averaged across the 8 assays, the lower limit of detection (B0 – 3 x SD method) was 0.1 ng/ml for cortisol assays and 3.9 pg/ml for progesterone assays. Average recovery values for external controls (Lyphocheks) were 90.2 and 90.8% for low and high concentration in progesterone assays, and 119.5 and 119.1% for low and high concentration cortisol controls.\n\nData analysis was performed using SYSTAT 13 and SPSS 21. Where raw hormone data are presented, salivary cortisol concentrations are reported as ng/ml and progesterone concentrations as pg/ml. To examine the overall magnitude of hormonal response to the tasks, we calculated the area under the curve with respect to increase (AUCi; Pruessner et al., 2003) from cortisol and progesterone Sample 3 (baseline/pre-task) to Sample 6 (post-task, at the end of the study). Sample 3 was chosen as the baseline as stress hormones are well-known to be elevated at the beginning of study sessions, owing to the novelty of the test environment, among other factors (see e.g. cortisol data in Abercrombie et al., 2006; further explanation in Wirth et al., 2011). Notably, AUCi calculations improve on difference scores because they utilize information for all measurements from Sample 3 to Sample 6. Previous studies have shown that cortisol tends to be elevated for up to 90 minutes after the TSST (e.g., Kirschbaum et al., 1995), so Sample 6 is timed appropriately to capture the end of most hormonal responses to the task. Therefore, the chosen number of samples and the timeframe used to calculate AUCi were selected to capture the complete cycle of hormonal change in response to the stressors/tasks.\n\nTo test our hypotheses about effects of the manipulations, as well as to test for sex differences, we first conducted an ANOVA on AUCi for the entire sample, with Group (TSST stress, TSST control, Cyberball rejection, or Cyberball control) and Sex as the independent variables. Second, to further explore how the effects emerge for each sex, we split the sample by sex and conducted ANOVAs for each sex on AUCi by group. Post-hoc Tukey tests were then used to follow up on all ANOVAs.\n\nMenstrual phase could be expected to impact hormone levels, particularly progesterone. Fortunately, by using AUC, initial differences in progesterone due to variations in menstrual phase are controlled for, since AUCi reflects the total amount of increase in the hormone from baseline — in other words, baseline differences are factored out. Furthermore, there was no correlation between progesterone AUCi and self-reported number of days since the start of the last menstrual period, i.e. the point that each woman was in her cycle (r2 = -0.077, p = 0.57). Neither was this relationship significant for cortisol AUCi (r2 = -0.073, p = 0.59). Also, self-reported days since period, entered as a covariate, did not moderate the effect of Group on either cortisol AUCi or progesterone AUCi in women. Therefore, for the purposes of the present research, we conducted analyses collapsing over menstrual phase. To more directly address the question of how menstrual phase impacts hormonal responses to tasks like the TSST and Cyberball, research would be needed selecting women in particular cycle phases; this was beyond the scope of the present report.\n\n\nResults\n\nParticipants who completed Cyberball, in both the inclusion and exclusion conditions, completed a questionnaire afterwards rating a number of statements regarding their inclusion and feelings during the game (Williams et al., 2000). T-tests were used to compare participants’ ratings on these items in the inclusion (control) vs. exclusion (stress) condition. As expected, participants in the exclusion condition rated that a smaller percent of the throws were made to them, and that the other game-players included them less, as well as excluded them more. They were less likely to endorse that they made a connection or bonded with one or more of the other game-players, and they rated themselves as feeling more like an outsider, more non-existent, and less in control. They rated themselves as feeling less able to throw the ball as often as they wanted, and less that their performance had any effect on the direction of the game. They also were significantly more likely to endorse that the other game-players failed to perceive them as worthy and likeable people (all p < 0.05). Excluded participants also endorsed at marginally greater rates the statement “I felt somewhat inadequate during the Cyberball game” (p = 0.055). There were no significant differences between the exclusion and inclusion groups on statements regarding feeling frustrated, angry, good about oneself, enjoyment of the game, or “felt as though my existence was meaningless” (even though excluded participants did rate “I felt non-existent during the game” significantly higher than included participants). Therefore, participants were clearly aware of the exclusion and had negative feelings about it. As mentioned above, when given an opportunity to give comments or observations about the study, no participants expressed suspicion that the other game-players were not real people.\n\nAn ANOVA with factors Group and Sex yielded a significant main effect of Group on cortisol AUCi, F(3,130) = 4.54, p = 0.005, partial η2 = 0.100. Neither the main effect of Sex nor the interaction was significant (main effect of sex: p = 0.188; interaction: p = 0.698). As expected, cortisol AUCi was highest in the TSST Stress group: M (SD) = 9.26 (32.67), compared with -2.59 (26.72) for TSST Control; and -16.41 (37.44) and -4.25 (16.52) for Cyberball Rejection and Control, respectively. Post-hoc Tukey HSD tests by Group revealed a significant pair-wise comparison only between TSST Stress and Cyberball Rejection groups (t(71) = -3.12, p = 0.003; 95% CI: -42.094 to -9.260, Cohen’s d = -0.731). However, as seen by the mean AUCs, the TSST Stress group was the only group with a positive AUCi, reflecting an overall increase in cortisol over the session.\n\nIn exploratory, separate ANOVAs conducted in women and men, Group significantly impacted cortisol AUCi in men (F(3,69) = 3.86, p = 0.013, partial η2 = 0.149). Post-hoc Tukey tests in men again revealed a significant pair-wise comparison between TSST Stress and Cyberball Rejection (t(35) = -2.70, p = 0.011; 95% CI: -43.53 to -6.18, Cohen’s d = -0.883) as well as a significant comparison between TSST Stress and TSST Control (t(32) = 2.07, p = 0.046; 95% CI: 0.325 to 37.656). In women, though again the highest and only positive cortisol AUCi was in the TSST Stress group (M (SD) AUCi = 3.23 (31.17), vs. -0.823 (40.23) in TSST Control; -23.65 (48.97) in Cyberball Rejection; -6.89 (15.49) in Cyberball Control), the ANOVA in women failed to reach significance (F(3,60) = 1.82, p = 0.154, partial η2 = 0.087). See Figure 2.\n\nSalivary cortisol for: entire sample, (a); men only, (b); and women only, (c). TSST = Trier Social Stress Test. CB = Cyberball. Ng/ml = nanograms per milliliter. Error bars indicate standard error of the mean.\n\nIn sum, TSST and Cyberball do not have the same effects on cortisol levels. The TSST Stress condition was the only condition which caused an increase in cortisol. Sex did not moderate this finding; however, when the sample was split by sex in an exploratory analysis, only in men did the effect remain significant.\n\nAn ANOVA conducted on progesterone AUCi with factors Group and Sex yielded no significant main effects or interactions, all p > 0.4. Neither were there any effects of Group on progesterone AUCi when examined separately in men or in women. Interestingly, in men, the Cyberball Rejection condition elicited the highest average progesterone AUCi out of the four groups; mean AUCi in men in Cyberball Rejection was 60.04 (229.55), vs. -45.20 (293.55) in Cyberball Control, and -69.37 (425.20) and 14.02 (353.24) in TSST Stress and Control, respectively. However, pairwise post-hoc comparisons failed to reach significance. Thus, there were no effects of either Cyberball or TSST on progesterone in either sex; see Figure 3.\n\nSalivary progesterone for: entire sample, (a); men only, (b); and women only, (c). TSST = Trier Social Stress Test. CB = Cyberball. Pg/ml = picograms per milliliter. Error bars indicate standard error of the mean.\n\n\nDiscussion\n\nThis study evaluated the effects of two different stress tasks, and their respective controls, on cortisol and progesterone. We found support for our first and second hypotheses, in that the TSST elicited a significantly greater cortisol response than all other tasks. Cyberball exclusion/social rejection was not associated with cortisol reactivity. This set of findings is in line with the physiological functions of glucocorticoids, which include mobilizing energy (Nelson, 2005; Sapolsky, 2002; Wirth & Gaffey, 2013). Cyberball exclusion is certainly unpleasant for participants (Williams et al., 2000; Zadro et al., 2004), but it is not a situation that demands or even allows very much active thought, planning, or physical activity. This is in contrast to the TSST, in which participants are continually actively modifying their speech in response to the feedback (or lack thereof) from the judges. The performance aspect of the TSST possibly requires more energy consumption by both the brain and body, and therefore a higher glucocorticoid response compared with Cyberball, which involves simply sitting at a computer pressing keys to determine the direction of the next ball toss.\n\nThese findings also underscore the fact that not every situation involving social rejection and associated negative feelings engenders a cortisol response, as well as the lack of a one-to-one relationship between negative feelings/mood/affect and cortisol. There are many examples of conditions in which cortisol is elevated without necessarily any changes to mood or affect, including exercise and illness. There are also examples of laboratory stimuli which sharply increase negative affect without affecting cortisol levels, such as viewing unpleasant pictures (Wirth et al., 2011; Wirth & Gaffey, 2013). Furthermore, meta-analyses across laboratory stressors show small or zero correlations between cortisol and subjective emotional responses (Campbell & Ehlert, 2012; Dickerson & Kemeny, 2004; Page-Gould et al., 2013).\n\nThe greater cortisol response to the TSST is also in line with Dickerson & Kemeny’s (2004) demonstration that social-evaluative judgment is the key factor in generation of cortisol responses in psychological laboratory tasks. Cyberball might be thought of as including social judgment, but there is very little for the other “players” to judge about the participant. In fact, in Cyberball exclusion, it is completely ambiguous why the other players cease throwing the ball to the participant. In the TSST, on the other hand, the constant monitoring and interruptions of the judges, along with their flat affect, can be taken by a participant to directly relate to their speech and arithmetic performance in real time.\n\nThese findings have implications for understanding the health consequences of real-world loneliness and social rejection. It is often speculated that HPA axis activity, specifically higher cortisol levels, might mediate the connection between social rejection and poorer health. However, at least in a laboratory setting, an acute social rejection experience does not cause a cortisol response, suggesting other mechanisms. Alternately, it may be that HPA activity only plays a role in chronic or “real-life” rather than acute, laboratory experiences of social rejection, e.g. loneliness (Adam et al., 2006; Hawkley & Cacioppo, 2010). Cyberball may not be the ideal task to study social rejection in the laboratory in relation to detrimental effects on health.\n\nIn contrast with our cortisol results, neither the TSST nor Cyberball induced a change in progesterone. This finding is somewhat surprising in light of research demonstrating that progesterone does increase in response to some types of stress (Childs et al., 2010; Wirth, 2011), including social rejection (Maner et al., 2010; Wirth & Schultheiss, 2006). Also, though there are prior reports that progesterone and cortisol levels increase and decrease in tandem in men and in women taking hormonal contraceptives – indicative of progesterone increasing alongside cortisol during stress - this was not found in cycling women (i.e., women not on hormonal contraceptives, such as in the present study; Wirth et al., 2007). Possibly, progesterone only increases during certain kinds of stressors, such as those including physical pain/distress (Wirth, 2011), or only under certain conditions, such as the morning (Childs et al., 2010). Another possibility is that, in social rejection contexts, progesterone responses are driven by a “tend-and-befriend”, affiliative response (Wirth, 2011). Though it creates a sense of rejection, the lack of face-to-face contact might cause Cyberball to not generate affiliative motivation to the same extent as other rejection tasks, or even film clips (Wirth & Schultheiss, 2006). Further research is necessary to comprehensively chart under what circumstances and what types of stressors cause increases in progesterone in humans. It is also important to characterize the conditions that provoke increases in downstream hormones like allopregnanolone, since allopregnanolone and related progesterone-derived neurosteroids could be important components of stress regulation (Wirth, 2011).\n\nSeveral limitations in this study should be acknowledged. Logistics of running the study demanded a lack of precise control over what menstrual phase the women participants were in. As mentioned above, however, a self-report measure of menstrual phase did not correlate with AUCi for either hormone and did not moderate any of the findings. A second potential limitation is sample size; though data from 131 participants was collected, power may still have been insufficient to detect small effect sizes that are commonly found in hormone-behavior studies. Furthermore, although every effort was made to conceal information about condition/group assignment from the participant until directly before their task, the study was only single-blind, and it is conceivable that the experimenters unconsciously treated stress versus control participants differently prior to the experimental manipulation.\n\nIn conclusion, we found evidence that, unlike a standardized speech task, Cyberball social rejection is not associated with a cortisol response in a sample of college students, despite feelings of rejection and exclusion engendered by this task. This evidence underscores the fact that the HPA axis does not have a one-to-one relationship with social rejection experiences and associated feelings. We also found a lack of evidence for a progesterone response to the cortisol-provoking speech stressor, as well as to the Cyberball rejection task. Taken with past work (Childs et al., 2010; Maner et al., 2010; Schultheiss et al., 2003; Wirth & Schultheiss, 2006; Wirth, 2011), these findings present a mixed picture in terms of evidence for progesterone responsivity to stress (and specifically to social rejection) in humans. Future work is needed to delineate precisely the types of emotional and social manipulations and physical stressors which lead to progesterone increases, as well as downstream neurosteroids. This work is important both from the perspective of basic physiology and psychology research, to understand the hormonal effects of stress and emotion in human beings, and also from a health standpoint, to better understand the mechanisms underlying impacts of stress and social rejection on human health.\n\n\nData availability\n\nfigshare: Cortisol and progesterone data collected in participants exposed to speech and rejection tasks. doi: 10.6084/m9.figshare.1150167 (Gaffey & Wirth, 2014).", "appendix": "Author contributions\n\n\n\nAG and MW together conceived the idea for the study and designed the experiment. AG carried out the research, conducted data analysis, and wrote the initial draft of the manuscript. AG and MW together completed additional writing, edited and finalized the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was funded in part by discretionary funds to Michelle Wirth from the University of Notre Dame. An NSF Graduate Student Fellowship supported Allison Gaffey during data analysis and manuscript preparation.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors would like to thank undergraduate research assistants Christina Buchanan, Kelsey Christoffel, Cristina Kline-Quiroz, Brandy Martinez, Kelly Miller, Kathleen Poplowski, and Mark Tancredi for their assistance with data collection. We thank Brandy Martinez for her assistance with coordinating research assistants, recruitment, data processing, and hormone assays. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "6019", "date": "29 Sep 2014", "name": "Richard Slatcher", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI enjoyed reading this paper and think that it will make an important contribution to the literature. This is one of those instances in which a null finding can potentially be very informative in terms of theory development. Although there are many different possibilities of why Gaffey & Wirth did not find effects of rejection on cortisol, the theory that they put forth—that rejection does not necessitate high energy mobilization needs compared to the acute stress of giving a speech—is an interesting and compelling one. Those of us who study cortisol often mention the energy mobilization aspects of cortisol as almost an afterthought. This paper puts the idea of energy mobilization front and center. Self-evaluative threat is clearly a key part of why the Trier Social Stress Test (TSST) typically “works” to raise cortisol levels. This paper suggests that social evaluative threat itself may be not sufficient for triggering a cortisol response, but rather that high energy mobilization may be necessary as well.Although I generally really liked this paper and thought that it was well written, I do have some concerns and suggestions, which I hope that the authors will find useful in revising this paper.In the 'Introduction', it is said that high levels of glucocorticoids have been proposed as one mechanism mediating the links between isolation and poor health. I think that perhaps the term “cortisol dysregulation” or “HPA axis dysregulation” might be better than “high levels.” The evidence that cortisol responses during the TSST are associated with poorer health is very slim, as are the data linking total cortisol output over the day (both AUCg and AUCi) to physical health. There is some evidence that flatter diurnal cortisol slopes (a less steep decline in cortisol across the day) are associated with poorer physical health/mortality, but the picture is clearly incredibly complex, potentially involving glucocorticoid resistance and many other intervening factors. The idea that higher cortisol is bad is, I think, overly simplistic; “dysregulation” would more accurate. Is loneliness the same as rejection? These two terms are used almost interchangeably in the Introduction, but are quite different. One can feel lonely without being rejected. Being rejected can lead to feelings of loneliness, but so can many other social conditions (not living near people, not going out much, being shy, etc.). I think it is important to make distinctions between loneliness and rejection/ostracism in the paper. Also, to what extent might chronic loneliness drive HPA activity compared to fleeting loneliness? Is it perhaps only chronic loneliness that really matters? Adam et al.’s 2006 PNAS paper looking at day-to-day loneliness and cortisol might be informative here. My guess is that most readers will be less familiar with the progesterone literature than the cortisol literature, so I think a little bit more thorough description of progesterone and its health and psychological correlates would be helpful. It is said in the 'Introduction' that there are important implications of progesterone “for understanding psychological disorders.” A couple of specific implications would be helpful there. In the paragraph following that one, it was quite unclear to me whether elevated progesterone is good or bad for one’s health (or neither, or unknown). For example, the statement “links progesterone to the beneficial effects of helping behavior on cardiovascular recovery from stress” makes it sounds like higher levels of progesterone are good. However, here and elsewhere in the paper, the direction of association (positive or negative) is omitted. Please go through and add in the direction of association throughout the paper, just so the reader is clear about which way the direction of effects are going. Another example “progesterone and not cortisol showed (positive?) associations with affiliation.” Were a priori power analyses conducted? If so, please report. If not, please report achieved power. Was self-reported loneliness and the subjective feeling of being rejected assessed after Cyberball? “I felt non-existent during the game” seems related to feeling rejected, but it is not the same thing. A concern that I had was whether or not Cyberball is a powerful enough rejection manipulation to elicit a cortisol response in the lab. To confidently refute the hypothesis that rejection leads to cortisol increases, I think we need to first know that the participants felt strongly rejected and/or lonely afterwards. I realize that Cyberball is a widely used paradigm, but it still may not be powerful enough to effectively test the rejection-cortisol association if people are not feeling powerfully rejected. Related to this, it is said in 'Cyberball manipulation check' that “participants were clearly aware of the exclusion and had negative feelings about it.” Clearly they were aware of the exclusion, but the results presented do not indicate that participants felt bad about it. Please rephrase/clarify. I think a great point of discussion could center around the idea of energy mobilization. If energy mobilization is key to why the TSST raises cortisol and why Cyberball doesn’t, then how might energy mobilization be directly tested? As I said at the beginning of my review, this is an exciting and provocative idea that could potentially be directly tested. How might one go about doing that? Looking at pre- and post-task depletion and/or fatigue? Changes in blood glucose? Some more discussion of this idea and steps forward would be a nice addition to the paper.", "responses": [ { "c_id": "1034", "date": "16 Oct 2014", "name": "Michelle Wirth", "role": "Author Response", "response": "We agree completely. We have made changes to the second paragraph of the Introduction to reflect the fact that all kinds of dysregulation in the HPA axis – including, as you mention, flattened/blunted diurnal patterns of cortisol – have been associated with poorer health. The changes we have made are subtle, however, because we stand by the research cited which found associations between (a) loneliness with higher cortisol, (b) social isolation with robust glucocorticoid increases in laboratory animals, and (c) chronically high cortisol with poorer health outcomes (not to say that is the only type of HPA dysregulation associated with health issues). We acknowledge at the end of this paragraph that these relationships are complex, and poorly understood, which I think also underscores your point. Loneliness vs. rejection: This is an excellent point, and though we do not believe these two conditions are identical or interchangeable, we do believe they involve similar kinds of subjective feelings, and also may share some physiological consequences (i.e., HPA axis activation). We have rewritten the second paragraph of the Introduction to be clearer that we are not conflating the two concepts, but that they are associated. We have also hinted at the need to study both acute rejection and long-term/chronic rejection or other forms of social isolation, although this is touched on more directly in the Discussion, where we are citing Adam et al. (2006), along with the possibility that HPA axis effects might only be seen in chronic rejection or isolation rather than fleeting feelings in laboratory studies. I hesitate to discuss the Adam et al. paper in more depth here, since those authors were examining diurnal cortisol profiles and cortisol awakening response, which is not necessarily comparable to response to a laboratory stressor. However, we do note that Adam et al. found that subjective loneliness associated with next-day cortisol – not same-day – suggesting longer-term effects. Unfortunately, we do not have next-day cortisol awakening response data in the current study. Progesterone / detail and directions of associations with health: Excellent point, and we would argue that to some extent, your point in #1 applies here as well, that it is likely dysregulation in progesterone / allopregnanolone rather than overall higher or lower levels that are associated with health issues. That said, there is evidence that psychological disorders as diverse as depression, PTSD, and schizophrenia are associated with lower levels of allopregnanolone compared with healthy controls (see Wirth 2011, Frontiers in Endocrinology). Progesterone, as the precursor for allopregnanolone, does not always show such differences; some evidence points to dysregulation in the enzymes needed to produce allopregnanolone from progesterone in psychopathologies, rather than differences in progesterone levels. However, measuring progesterone changes in response to social rejection and other stressors is relevant since progesterone levels are one factor (along with enzyme availability) that helps determine levels of neurosteroids such as allopregnanolone. As requested, we have added an example in the introduction (lower allopregnanolone in depression), and we have clarified the association between progesterone and affiliation in the sentence noted. Unfortunately, we did not conduct a priori power analyses. We do now report achieved power, however. As per our response to Jens Pruessner, the other reviewer: We performed a post-hoc power analysis, calculated using the partial eta squared obtained in our omnibus ANOVA of .10, and found that, with our sample size of 131, we had power of .90 to detect an effect of this size. Therefore, we believe we had adequate power to have detected even relatively small effects. We unfortunately do not have data specifically on how rejected participants felt after Cyberball. The question appeared ambiguously on the questionnaire we administered: It was worded “How accepted/rejected did you feel?”, without explicit instructions to circle or indicate “accepted” vs. “rejected” – which most participants did not do, making their numerically rated responses uninterpretable. However, we would argue that from the post-Cyberball “manipulation check” questionnaire, participants not only were aware of the exclusion, but also had negative feelings. For example, as stated in the Results, participants in the Cyberball exclusion condition rated themselves as feeling more like an outsider, more nonexistent, and less in control, compared to the Cyberball inclusion condition. These are arguably negative feelings, which were increased in the exclusion condition. Exclusion condition participants also scored lower on ratings of the other game-players perceiving them as worthy and likable people, which, while not a direct report of feelings, can reasonably be expected to be associated with feelings of rejection. However, it is true (and we state in the results) that there was no difference in particpants’ feelings of frustration or anger. We agree that Cyberball does not seem like a very powerful manipulation, that there may exist other social rejection manipulations might elicit more powerful feelings of rejection, and possibly also changes in cortisol or progesterone. We address this in the Discussion. Directly testing energy utilization is an excellent idea – however, to accomplish this in practice could be logistically quite difficult. To accurately measure energy usage, participants are typically tested for several hours in sealed chambers so that all their oxygen consumption and carbon dioxide output can be measured (i.e. calorimetry). Overnight stays are typically used to calculate basal metabolic rate, as for accurate measurement the sympathetic nervous system must not be activated due to novelty of the test environment, etc. let alone laboratory stressors. I don’t think blood glucose measurements would be an accurate way to measure energy consumption during stressors, since as glucose is depleted for energy during the stressor, it will be rapidly replenished by glucocorticoids, making interpretation of changes difficult. There are kinesiology and exercise physiology laboratories studying energy usage in humans, so one possible step forward might be a collaboration between e.g. kinesiologists and psychologists. However, one barrier is that these two groups are interested in very different research questions. For these reasons, directly measuring energy utilization is perhaps not an ideal first step forward for this line of research." } ] }, { "id": "6015", "date": "06 Oct 2014", "name": "Jens Pruessner", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper by Gaffey and Wirth pursued two main goals. One, to identify whether 'Cyberball', a frequently used task in the Social Neurosciences, elicits a significant cortisol response (and how that compares to the well described cortisol stress response of the Trier Social Stress Test, TSST). Two, to determine whether these stress tasks also elicit a significant change in circulating levels of progesterone, as previous research provided some evidence that progesterone might respond to a psychosocial stress task as well.The authors report that there was no increase in cortisol to the 'Cyberball' task (although there was the expected increase in response to the TSST), and they further did not observe any significant change in progesterone levels in response to either task.There are many positive things to be said about the study. The experimental design is innovative, and the major research questions (both relating to the stressfulness of Cyberball and the responsivity of progesterone to stress) are original. The combination of the two goals into one study is actually an added plus, as this allowed the cross-validation of a possible progesterone response to a stressful task using two different experimental manipulations.The study is also nicely powered (n=131), although not completely balanced. The obvious main drawback of the study is the lack of significant results; unfortunately, the authors could not find any evidence for a progesterone response to stress, and no evidence for a cortisol response to stress. As with any negative finding, it would be important to actually estimate the expected effect size from previous studies and use that to compute the power for the current study - in other words, what were the chances of finding a significant effect given the sample size present in this experiment? That information would allow the reader some judgment as to whether no significant changes are to be expected even if larger sample sizes were to be assessed.Related to this comment, the advantages and disadvantages of a between-subject versus within-subject design could also be discussed; since Cyberball and the TSST are conceptually and practically two very different tests, a within subject design could be envisioned where subjects are exposed to both the TSST and Cyberball in a counterbalanced manner, perhaps with one week difference. The net investment in time and effort would probably have been very similar, but the added advantage would have been to compare results in cortisol and progesterone within the individual (which might not have made a difference in case of insignificant stress responses to Cyberball, but still). This is not really a critique but rather a comment that could be entered into the discussion, to alert the reader to the possibility of alternative experimental designs.Another comment relates to the menstrual cycle phase, which the authors mentioned they couldn't control for in the current study. This is if course suboptimal and the authors already acknowledge this in the limitation section; however, they argue that the damage is minimal as they were interested in the change from baseline, rather than baseline differences, thus the chosen measure (AUCi) can compensate for the lack of menstrual cycle phase control. I am not convinced that this approach really resolves this particular problem - what if the magnitude of the change depends on the baseline level? For example, if you are already high at baseline, you won't see a strong response anymore? Even though there is no evidence for this in the current study, I would not be comfortable with accepting the advise that it is OK to not control for menstrual cycle phase when measuring progesterone levels in cycling young women.One other aspect of the experimental design caught my attention - the authors mention that they used the essay writing about the dream job (which is part of the TSST control condition) to fill up the time difference between the Cyberball and the TSST sessions. While it is commendable to control for total time exposure, the authors have now in reality confounded the Cyberball with at least part of the TSST control condition - might that have induced some interaction effects? From Figure 2, it appears that the TSST control (TSST-C) is actually leading to a less pronounced circadian decline overall, and that the cortisol increase between sample 3 and 4 in the group of women is actually strongest in the TSST-C group. So by itself, the TSST-C might have some effects on at least cortisol; combining it with the other stress task you want to compare against might then present a suboptimal approach, and should be critically discussed as well.On other point: In the past, effects from the biological sex of the TSST panel on the magnitude of the cortisol stress response in the test subjects has been observed. The paper doesn't mention the sex of the judges - were those mixed (one man, one woman), or unisex (which?), or changing depending on availability? For the Cyberball, what was the sex of the other 'players'? Depending on the setup, this should either be evaluated, or mentioned as a possible limitation as it could explain additional variation in the endocrine data.Finally, one technical question: What was the reason for the three subsequent freeze-thaw cycles prior to performing the assay? Was that a recommendation by the manufacturer? This information should be added to the methods, rather than just stating it.Overall, I think that this study is an important contribution to the literature. The observation that Cyberball might not cause an increase in cortisol is important for many stress researchers contemplating various experimental designs but for that reason the addition of a power calculation is essential.", "responses": [ { "c_id": "1033", "date": "16 Oct 2014", "name": "Michelle Wirth", "role": "Author Response", "response": "We agree that a power analysis is necessary in order to have confidence in these null findings. We performed a post-hoc power analysis, calculated using a partial eta squared of .10 (the small effect size we obtained in our Group X Sex ANOVA on cortisol), and found that, with our sample size, we had power of .90 to detect this effect. Therefore, we believe we had adequate power to detect even quite small effects. Within-subject designs: although this was a possibility, there would have been several disadvantages to performing this study using a within-subjects design, including habituation and order effects. It is well-known that participants quickly habituate in their cortisol responses to the TSST. Laboratory stressors might show cross-habituation, so that null effects in subsequent sessions would be ambiguous: are they due to habituation, or because those conditions do not produce hormonal responses? In general, order effects are common in within-subject hormone research (e.g. Wirth, Scherer, Hoks & Abercrombie, 2011, Psychoneuroendocrinology; Herzmann, Young, Bird & Curran, 2012, Brain Research). With four conditions (since the TSST and Cyberball have their own, separate control conditions) we would have to potentially consider the effects of 16 different orders of conditions- which is prohibitive logistically and also would have reduced our power. This is why for this study, we administered conditions between subjects. Within-subject designs are certainly a viable option for other kinds of studies, but did not seem practical or appropriate in this case. Menstrual phase: we agree that the magnitude of change in a hormone could depend on baseline levels. This is why we did conduct our analyses with menstrual phase (“days ago” variable) as a covariate. See third paragraph of Data Analysis: “self-reported days since period, entered as a covariate, did not moderate the effect of Group on either cortisol AUCi or progesterone AUCi in women.” This test would show us if the magnitude of the change in progesterone, as represented by AUCi, was affected by baseline progesterone levels, as represented by days since period. However, we agree that this is a suboptimal approach, since days since last period is a suboptimal method of assessing menstrual phase. We agree that future research assessing progesterone levels in women should more carefully control for menstrual phase. We have added a recommendation to this effect in the Limitations, under Discussion. Writing task as potential confound: first, in case there is any confusion, an important difference between the writing task in the Trier control and Cyberball versus the Trier stress condition is that, in the Trier stress condition, participants knew that they are taking notes for a speech they would have to give. In the other two conditions, participants were told that the essay’s content would not be judged or evaluated in any way. As for overlap between the Trier control and the Cyberball conditions, writing an essay under these instructions has been used in Trier control conditions frequently in our and other laboratories (e.g. our colleague Jessica Payne; see also Het et al. 2009, Psychoneuroendocrinology). Having to write about one’s dream job when one is told the writing will not be evaluated or judged does not seem to elicit any cortisol response. Therefore, in the Cyberball condition, we expected that any response would be due to the Cyberball rejection manipulation, not due to the writing task. In figure 2C, the greatest increase between time point 3 and time point 4 in women is actually in the Trier stress group (dotted line with black squares is the Trier control group), although the error bars are overlapping for these two groups such that there are no significant differences. The panel of judges always consisted of one man and one woman; we agree that this is important information and have added this to the Methods, under Tasks. In Cyberball, the other “players” were always both of the same sex as the participant. This was already mentioned in passing under Tasks, however we have now made it more explicit. Freeze-thaw cycles are part of our standard processing steps for saliva samples in order to break up long-chain mucopolysaccharides, and thereby make the saliva less viscous and able to be pipetted accurately. See e.g. Wirth and Schultheiss 2006, Hormones and Behavior. This information has been added to the hormones section of the Methods." } ] } ]	1	https://f1000research.com/articles/3-208
https://f1000research.com/articles/3-258/v1	29 Oct 14	{ "type": "Research Article", "title": "Methodological framework to identify possible adverse drug reactions using population-based administrative data", "authors": [ "Brian C. Sauer", "Jonathan Nebeker", "Shuying Shen", "Randall Rupper", "Suzanne West", "Judith A. Shinogle", "Wu Xu", "Kathleen N. Lohr", "Matthew Samore", "Jonathan Nebeker", "Shuying Shen", "Randall Rupper", "Suzanne West", "Judith A. Shinogle", "Wu Xu", "Kathleen N. Lohr", "Matthew Samore" ], "abstract": "Purpose: We present a framework for detecting possible adverse drug reactions (ADRs) using the Utah Medicaid administrative data. We examined four classes of ADRs associated with treatment of dementia by acetylcholinesterase inhibitors (AChEIs): known reactions (gastrointestinal, psychological disturbances), potential reactions (respiratory disturbance), novel reactions (hepatic, hematological disturbances), and death.Methods: Our cohort design linked drug utilization data to medical claims from Utah Medicaid recipients. We restricted the analysis to 50 years-old and older beneficiaries diagnosed with dementia-related diseases. We compared patients treated with AChEI to patients untreated with anti-dementia medication therapy. We attempted to remove confounding by establishing propensity-score-matched cohorts for each outcome investigated; we then evaluated the effects of drug treatment by conditional multivariable Cox-proportional-hazard regression. Acute and transient effects were evaluated by a crossover design using conditional logistic regression.Results: Propensity-matched analysis of expected reactions revealed that AChEI treatment was associated with gastrointestinal episodes (Hazard Ratio [HR]: 2.02; 95%CI: 1.28-3.2), but not psychological episodes, respiratory disturbance, or death. Among the unexpected reactions, the risk of hematological episodes was higher (HR: 2.32; 95%CI: 1.47-3.6) in patients exposed to AChEI. AChEI exposure was not associated with an increase in hepatic episodes. We also noted a trend, identified in the case-crossover design, toward increase odds of experiencing acute hematological events during AChEI exposure (Odds Ratio: 3.0; 95% CI: 0.97 - 9.3).Conclusions: We observed an expected association between AChEIs treatment and gastrointestinal disturbances and detected a signal of possible hematological ADR after treatment with AChEIs in this pilot study. Using this analytic framework may raise awareness of potential ADEs and generate hypotheses for future investigations. Early findings, or signal detection, are considered hypothesis generating since confirmatory studies must be designed to determine if the signal represents a true drug safety problem.", "keywords": [ "adverse drug reactions", "adverse drug events", "drug safety", "patient safety", "Medicaid", "pharmacoepidemiology", "post-marketing surveillance", "propensity scores", "acetylcholinesterase inhibitors" ], "content": "Introduction\n\nDespite its limitations, the Food and Drug Administration’s (FDA) Adverse Drug Event Reporting System (FAERS) has successfully identified rare and unexpected adverse events1–3. In many previous studies, administrative data sources have been used to estimate the extent of the problem or confirm safety signals identified from AERS4,5. However, fewer studies have demonstrated the potential of administrative data for first-line adverse drug reaction (ADR) surveillance6. In this pilot study, we present a framework for directed discovery of possible ADRs using population-based administrative data sources, an approach intended to complement the FDA’s adverse reporting system. We describe our approach as directed because we target specific health outcomes of interest instead of simply mining the data for statistical associations.\n\nWe examined the associations between drug use and possible ADRs resulting from treatment of dementia with acetylcholinesterase inhibitors (AChEIs), namely, donepezil hydrochloride, rivastigmine tartrate, and galantamine hydrobromide. We measured associations for four classes of ADEs—established reactions (gastrointestinal and psychological disturbance), potential reactions based on drug pharmacology (respiratory disturbance), novel unexpected reactions (hepatic and hematological disturbance), and death. Hepatic and hematologic syndromes were evaluated because they are two examples of potentially fatal reactions that have been found in post-marketing surveillance of drug-induced disease7.\n\n\nMethods\n\nThe directed discovery framework consists of clinical framing, data preparation, event detection, and hypothesis generating and testing. The first three components are described in the Methods; hypothesis generating and testing are explored in the Discussion.\n\nClinical framing consisted of reviewing the medical literature and consulting clinical experts to define the treatment groups, inclusion criteria, drug courses, outcomes and covariates.\n\nSources. Data consisted of pharmacy and medical claims and enrollment status from Utah Medicaid recipients in the fee-for-service program between 1/01/2003 and 12/31/2005. We linked Utah death-certificate data to Medicaid recipients by a deterministic method using a social security number. To protect patients’ privacy, all potentially traceable personal identifiers were removed. The University of Utah Institutional Review Board approved this study (IRB_00016984).\n\nSubjects. We studied Utah Medicaid recipients’ aged 50 and older with a dementia-type diagnosis (Table 1). As Medicaid enrollment occurs on a monthly basis, we tracked membership enrollment and de-enrollment and censored the patients whose enrollment was terminated and not re-established within the study period. Because of the relatively high rate of sustained enrollment, approximately 99% of the cohort was enrolled for at least 80% of the months from their first until their last month of eligibility or until the study period ended. We did not limit inclusion to continuously enrolled recipients.\n\nTreatment Groups. We inferred patient AChEI use by reconstructing courses of AChEI therapy from pharmacy claims data. To achieve a greater homogeneity among users’ disease stage and risk of adverse reactions8, we restricted the AChEIs cohort to the first incident course of AChEI therapy, which was defined as their first course with at least a 180-day drug-free period. To ensure that patients were receiving medical care during the 180-day drug-free period and were not receiving the drug elsewhere, recipients’ had to be enrolled and to have at least one medical claim during the 180-day drug-free (baseline) period. We defined a course of AChEI therapy as beginning on the week the drug was first dispensed and ending on day 60 after a continuous gap in the drug supply of ≥ 60 days (Figure 1).\n\nAChEI = Acetylcholinesterase inhibitors. Rx = Dispensed Prescription.\n\nThe untreated comparison group consisted of Medicaid recipients 50 years and older with a dementia-like diagnosis who did not receive AChEI therapy. We established a 180-day baseline period during which recipients were enrolled and had at least one medical claim. The index date for individuals in the untreated group began at the first dementia-related outpatient visit that allowed for a 180-day baseline period. Starting time zero with a dementia-related outpatient visit established an indicated population that was engaging the health care system.\n\nOutcomes. As noted earlier, our primary clinical outcomes were gastrointestinal, psychological, respiratory, hematological and hepatic conditions, and death. We identified health care visits related to each clinical outcome in professional and facility claims using Healthcare Cost and Utilization Project (HCUP) Clinical Classification Software (CCS) codes (documented in Table 1). As a primary diagnosis typically indicates the reason for seeking medical care or the most important problem at the visit, we limited the outcome detection to the primary diagnosis codes. We tailored outcome classifications for each study design (described under Event Detection). Our analysis also measured the association of AChEI use with death.\n\nPotential confounding. We assessed demographic variables, comorbidities, drug therapy, and indicators of health care utilization as potential confounders. Comorbidity indices included HCUP comorbidity software version 3.2 and the modified RxRisk-V (RxRisk-Vm) score, which infers comorbidity using pharmacy claims9. We measured health care utilization by considering the number of outpatient visits, hospitalizations, and emergency department (ED) visits, and we also accounted for use of hospice services and nursing home care.\n\nWe considered specific classes of medications as potential confounders—specifically, antianxiolytics, anticonvulsants, Parkinson’s treatment, antidepressants, antipsychotics, steroids, narcotics, respiratory agents, anticoagulants, corticosteroids, and sedatives. We treated the use of statin drugs as an indicator of health status because they are preferentially prescribed to healthier, less frail patients who are not at the end of life10.\n\nPerson time unit. We constructed the final analytic table using 1-week discrete time intervals; i.e., changes in covariate status, medication use and outcomes are captured weekly. This interval maximizes efficiency without omitting clinically important changes in patient outcome and covariate status. All database manipulation was conducted in SAS 9.2.\n\nCohort design. We used an open cohort design with propensity score matching to explore associations between data on drug utilization and possible ADRs. We used propensity scores to address covariate imbalance using logistic regression models to predict AChEI treatment. We included confounders and risk factors in the propensity score models11. Because we included risk factors along with confounders, we built separate propensity score models and matched cohorts for each study outcome. Two physicians who routinely treat patients with dementia independently selected variables to construct propensity score models. They discussed disagreements to arrive at consensus. Variables for each model are listed in Table 3.\n\nOur analyses included propensity score matching followed by additional matching on key prognostic covariates12. For example, we performed propensity matching with covariate matching whether an individual had a gastrointestinal visit during the baseline period when evaluating the gastrointestinal outcome. Analysis of death consisted of propensity score matching and covariate matching for baseline age and hospice care.\n\nClinical endpoints were intended to measure increased health care utilization associated with specific diagnoses. We defined episodes of care to differentiate clusters of events and to reduce the impact of immediate clinical exuberance associated with a new episode of care. A 4-week gap in claims for each clinical outcome was required to initiate a new episode. For each study endpoint, we calculated the incidence densities per 100 patient-years.\n\nWe established matched untreated cohorts using Mahalanobis metric matching13. Baseline characteristics of patients in the AChEI-treated and matched untreated cohorts were compared using Student’s t-tests and chi-square tests. We used conditional multivariable Cox-proportional hazard models that allowed for recurrent events to assess the effect of AChEI on specific clinical endpoints14. All statistical analyses were performed with Stata MP 9.2 for Windows.\n\nCase-crossover design. We established three 6-week time-windows (pre-treatment, first treatment, second treatment window) to assess acute and transient effects of AChEI treatment (Figure 1). The index week for the pre-treatment window was the week following the most recent clinic visit for any condition during the baseline period.\n\nTo capture acute effects of AChEI treatment, we used the week the AChEI was first dispensed as the index week for the first treatment window. We compared the odds of experiencing an event during that window with the odds of experiencing an event during the pre-treatment window to identify acute treatment effects. To evaluate the transience or stability of possible ADRs, we compared the odds of experiencing an event during the second treatment window to the odds of experiencing an event during the pre-treatment window. Patients were noted as having an event if they had a medical claim with the primary clinical diagnosis code of interest; we used only one event per time-window. Odds ratios between the referent and treatment windows were computed using conditional logistic regression. See Figure 2 for a summary of the two designs.\n\n\nResults\n\nOf the 29,046 eligible patients in the study populations, 4,109 had a medical claim with a dementia diagnosis between 1/01/2003 and 12/31/2005. The AChEI-treated cohort consisted of 976 total users and 332 users with incident courses; of the latter, 224 were started on donepezil, 59 on rivastigmine, and 49 on galantamine. Because the numbers of incident users of specific AChEIs were small, we did not assess potential ADRs for individual drugs. In the AChEI-treated group the median duration of incident courses was 33.4 weeks with an interquartile range (IQR) from 15 to 68.5 weeks. The median proportion of weeks for which the AChEI-treated group was estimated to have access to the medication at least 1 day during the week was 100%, with an IQR of 95%–100%. The untreated cohort consisted of 2,968 patients who were diagnosed with dementia but did not receive medication to treat the disorder during the study period (Figure 3).\n\nNote: Groups highlighted in blue met the inclusion criteria for this study and were the primary comparison.\n\nBasic characteristics of the study population during the 6-month baseline period are presented in Table 2. Compared with the untreated population, incident AChEI users were slightly younger, had fewer HCUP comorbidities, fewer clinic visits, and a lower frequency of hospice care. Incident AChEI users also had a higher frequency of statin use and nursing home care. RxRisk-Vm scores and the average numbers of hospitalizations and ED visits were similar for AChEI users and non-users (untreated patients).\n\nAChEI = Acetylcholinesterase inhibitors\n\nHCUP = Healthcare Cost and Utilization project\n\nSD = Standard deviation\n\n% = Percent\n\nED = Emergency department\n\nRxRisk-Vm = modified RxRisk-V\n\nAfter propensity score matching for each clinical endpoint, the two groups were similar on all variables for each outcome-based cohort, except the average number of ED visits, which was slightly higher in the untreated matched groups for the evaluation of respiratory and hepatic episodes (Table 3). In general, the lack of statistically significant differences between the AChEI-treated and untreated groups on propensity-adjusted variables suggests a balance in measured covariates between treatment groups.\n\n\n\np-value <0.05\n\nNo. = number\n\nTable 4 presents the incidence densities per 100-person years and 95% confidence intervals for the complete untreated population and propensity-matched comparisons. Table 5 presents the hazard rates for all unadjusted and matched comparisons.\n\nCrude analyses. In bivariate analysis (Table 5) we did not observe a higher rate of gastrointestinal episodes in the group treated with AChEIs compared to the untreated group. The rates of psychological episodes, respiratory episodes, hematological episodes, and hepatic episodes were slightly higher, but not statistically significantly, in the group treated with AChEIs compared to the untreated group. The rate of death in the group treated with AChEIs was significantly lower than in the untreated group.\n\nPropensity-matched analyses. We observed significantly higher rates of gastrointestinal episodes (Hazard Ratio [HR]: 2.02; 95% CI: 1.28 - 3.2) and hematologic episodes (HR: 2.32; 95% CI: 1.47 - 3.67) in the AChEI-treated group than in the propensity-matched untreated group (Table 5). For psychological episodes, respiratory episodes, and hepatic episodes, we observed higher, but not statistically significant, rates in the AChEI-treated group than in the propensity-matched untreated group. We observed a weak and non-significant association between AChEI treatment and mortality.\n\nCase-crossover analysis. In crossover analysis we did not observe increased odds of experiencing gastrointestinal events during either the first or second treatment windows. We observed an acute, but non-significant, effect of AChEI treatment on the odds of experiencing a psychological event during the first-treatment window; this was not sustained during the second-treatment window. We observed acute, but non-significant, effects of AChEI treatment on the odds of experiencing respiratory event and hematological events during the first-treatment window; both rates appeared to decrease during the second-treatment window. The acute effect of AChEI treatment on the odds of experiencing a hepatic event during the first-treatment window was imprecise and appeared to decrease during the second-treatment window (Table 6).\n\n\nDiscussion\n\nWe developed a cohort-based framework for using population-based administrative data to identify known ADRs and to discover ADRs that may have gone unnoticed during clinical trials. We evaluated AChEI therapy in people with dementia, considering a composite of possible ADRs─ i.e., expected, suspected, unexpected reactions, and death ─to demonstrate that our analytic techniques produced expected results. We used propensity score matching and a within-subject design in an attempt to handle confounding. Our pilot study examined data from patients diagnosed with dementia for both cumulative effects of AChEI treatment and acute effects following initiation of AChEI therapy. We demonstrated this approach with Medicaid data from the state of Utah; nonetheless, the framework presented here can be transferred for use with other health insurer databases, including the Medicare Parts A, B, and D data now available.\n\nA pervasive issue in pharmacoepidemiologic studies is confounding by indication15. This problem arises because factors that influence treatment choices made by clinicians also influence outcomes. Confounding by indication can bias the crude association between drug treatment and outcomes in either direction and with unknown magnitude. Propensity score models are one method used in pharmacoepidemiologic studies to balance measured confounders with the goal of making the treatment groups exchangeable.\n\nIn this study, we addressed confounding by indication by developing propensity score models for each study outcome. Theoretical confounders available in the data were included in each model to reduce bias. Before matching, the untreated group appeared to be frailer than the treated group; they had a higher proportion of hospice care, more comorbidity, and a lower proportion of statin users, which suggested less aggressive care because of poorer health. As one would expect, the unadjusted analysis made AChEI treatment appear protective against mortality when compared with the untreated group (HR: 0.66; 95% CI: 0.52 - 0.82), which is not supported by clinical trials or other observation studies16,17. After propensity and covariate matching we found no difference between the AChEI-treated and untreated groups (HR: 1.07; 95% CI: 0.74 - 1.54). This illustrates the importance of addressing confounding by indication when designing ADR surveillance systems.\n\nAn alternative approach to addressing confounding is to use inverse probability weighting (IPW) methods to model time-varying treatments and confounders. In simulation studies, these methods were less biased than conventional methods when time-varying confounding was present18. When allowing treatment to be time-varying, we observed gastrointestinal disturbance and discovered hematological disturbance; we noted the same findings as if follow-up began at initiation of drug treatment (data not shown). Future work should explore the presence of time-varying confounding and the benefits of using IPW methods to discover novel ADRs associated with drug treatments.\n\nTo evaluate possible acute and transient effects of AChEI treatments, we employed a type of case-crossover analyses. Typically in case-crossover analyses, events are compared between event and control time-windows for each individual. A major benefit of this within-subject design is that each person acts as his or her own control19,20. It also accounts for confounding by indication and other time-invariant and difficult-to-measure confounders. The drawback of such designs involves changes in treatment utilization that are influenced by health status or the study endpoints in question21. For example, when day-level drug utilization data are inferred from dispensing history, determining whether adverse effects are truly transient or the result of a decrease or discontinuation of drug treatment is difficult. Ultimately, we deemed the within subject analysis to be an excellent complement to the propensity score approach because of its ability to discover acute and transient effects and for its simplicity and ability to remove time-invariant confounding by indication.\n\n\nHypothesis generating and confirmation\n\nThe framework described here provides a structured approach for confirming expectations by evaluating known ADRs and discovering new ADR safety signals, such as the association we found between AChEI use and hematological disturbance. In support of the analytical effectiveness of these procedures, our approach confirmed an association with an expected reaction, gastrointestinal disturbances. The findings from the two study designs, however, were not consistent. Our inability to find an acute increase of gastrointestinal events in the first-treatment time window may be attributable to insensitivity of claims-based coding to identify symptoms of gastrointestinal disturbance.\n\nDespite the fact that our approach detected a significant association with one expected reaction, gastrointestinal disturbance, it failed to identify a strong positive association with the second expected reaction, psychological disturbance. We did show a higher rate of psychological episodes in the propensity-matched analysis; nevertheless, the association was not statistically significant. We did; nonetheless, observe higher odds of experiencing psychological events in the first-treatment time window than in the pretreatment time window using the within subject design. Even though the higher odds was expected, it was not statistically significance. This result can likely be attributed to a combination of factors. First, is the low power in the within subject design and second may be insensitivity of claims-based coding to identify symptoms of psychological disturbance.\n\nWe discovered no clear associations between AChEIs and respiratory disturbance or death. In a recent sequence symmetry analysis, the initiators of AChEI had no detectable increased rate of complications of chronic airway disorders25. We found no clear evidence of an increase or decrease in mortality associated with AChEI treatment in published studies or meta-analysis to which to compare our results16.\n\nOur analysis of unexpected reactions discovered a statistically significant positive association between AChEI treatment and hemotological episodes. Hematological events also appeared to be positively associated with early AChEI treatment. A detailed review of results with hematological event subcategories (not reported here) found that the rate of anemia was much higher in the AChEI-treated group than in the untreated group during the first 6 weeks of drug treatment. Further analysis is required to determine if this higher rate is causally associated with initiating anti-dementia drug treatment. At present, no known pharmacologic or empirical reasons can explain how AChEI drugs cause hematological toxicity.\n\nThe incidence of hepatic disturbance appeared to be higher in the treated group, although non-significant, in both the within subject and propensity matched design. Hepatotoxicity was a major safety concern with tacrine, which is the reason why it is no longer a commonly used; hepatotoxicity has not been reported for other AChEIs26. Larger observational studies are needed to determine whether an association between AChEIs and hepatotoxicity exists.\n\n\nLimitations\n\nThe results from this study are considered hypothesis generating rather than identifying causal treatment effects. Causal studies require validation of treatments, outcomes, and covariate classifications. Furthermore, causal studies require a stronger theoretical understanding and explication of the underlying causal relationships between the treatment and outcomes.\n\nWe compared AChEI-treated patients with an incident AChEI course of therapy, to an untreated cohort of patients with a dementia diagnosis. Other options were to compare directly the safety of AChEI products with one another or to compare the safety of AChEI therapy with the safety of other classes of medications used to treat such patients’ dementia. We were not able to compare individual drug products. Treatment with AChEIs is not directly comparable to treatment with memantine, a glutamaterginc N-methyl D-aspartate (NMDA) receptor antagonist, because memantine is typically not the first-line treatment for dementia; rather it is used in addition to an AChEI therapy, complicating any comparison.\n\nIn pharmacoepidemiologic studies, an untreated referent group can also be defined as patients with an incident course of a medication that is not associated with the indication or evaluated outcomes. This type of “active control group” is likely to be more similar to the treated group in regard to the activation of the health care system than the indicated but untreated group17. Drug dispensing indicates that the patient has activated the health system. In addition, prescription of a new medication is likely to result in closer monitoring and evaluation of an individual’s health status. The primary concern when comparing treated with untreated groups is under-recording of health conditions, making the members of the comparison group seem healthier than they really are, which can lead to overestimation of the effects of drug treatment.\n\nBecause of the multiple outcomes in this study, we were unable to identify a single medication that could yield comparable cohorts for all events. Instead, we used a dementia-related visit, not drug dispensing, as the index date for the untreated group. For both cohorts, the median amount of time to a clinic visit following the index date was 3 weeks, and the longitudinal visit process was also similar. These patterns suggest that health care access and followup may have been similar for the two groups.\n\nAnother limitation of this study is the small number of subject in the AChEI treatment group. This markedly limited our ability to confirm the expected adverse effects of AChEI treatment and discover adverse events that may have gone undetected in clinical trials.\n\n\nFuture research\n\nThe discovery of an association between a drug treatment and a theoretical reaction, an idiosyncratic reaction, or death is considered hypothesis generating or signal detection. Confirmation requires additional observational and possibly experimental studies. Ideally, discovered associations would first be confirmed or further characterized in large, disparate data sources to reproduce evidence of the association across different populations. In May 2008, the FDA published The Sentinel Initiative report to present the national strategy for monitoring medical product safety27. Their approach primarily establishes a nationwide health information network for confirmation of safety signals across multiple large databases. Additional observational studies along with richer clinical information such as electronic health records or prospectively designed studies, however, may be needed to characterize the causal relationship between a drug treatment and the adverse outcome.\n\n\nData availability\n\nThe raw data are available upon request. IRB approval and signed Data Use Agreements with the Utah Department of Health. For more information, please contact the corresponding author Brian Sauer.\n\n\nEthical considerations\n\nThe primary ethical consideration is the privacy and confidentiality of patient data. Limited datasets were used that restricted the use of direct patient identifiers. Data are stored on secured servers and only shared according to IRB policy and state data use agreements.", "appendix": "Author contributions\n\n\n\nConcept and Design: BCS, JRN, RR, MS\n\nAcquisition of Data: WX, BCS\n\nAnalysis and interpretation of data: BCS, JRN, SS, SLW, JAS, WX, RR, KNL, MS\n\nDrafting Manuscript: BCS, WX, JAS\n\nCritical Revision of Manuscript: BCS, SLW, KNL\n\nStatistical Analysis: BCS, SS\n\nObtaining funding: KNL, WX, MS\n\nAdministrative, technical or material Support: KNL\n\nSupervision: MS, KNL\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe original research was supported by an award from the Agency for Healthcare Research and Quality to RTI International, Contract No. HHSA 290 2005 0036 I. Brian C. Sauer was also funded by the Veterans Affairs Health Services Research and Development Career Development Award (RCD 06-300-2).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nThe authors thank the Utah Medicaid Program for providing the data and Zhiwei Liu for extracting the data and providing expert SAS consultation. We also extend our appreciation to Linda Lux, M.P.A., Jacqueline Amoozegar, B.A., and Loraine Monroe, all of RTI International, who provided dedicated support and assistance to the original project on which this work is based. The authors of this paper are responsible for its content. Statements in the paper should not be construed as endorsement by the Agency for Healthcare Research and Quality or the U.S. Department of Health and Human Services of a particular drug, device, test, treatment, or other clinical service.\n\n\nReferences\n\nBlum MD, Graham DJ, McCloskey CA: Temafloxacin syndrome: review of 95 cases. Clin Infect Dis. 1994; 18(6): 946–50. PubMed Abstract \| Publisher Full Text\n\nMedWatch Safety Reports. 2008. (Accessed 09/17/08, 2008). Reference Source\n\nWysowski DK, Swartz L: Adverse drug event surveillance and drug withdrawals in the United States, 1969–2002: the importance of reporting suspected reactions. Arch Intern Med. 2005; 165(12): 1363–9. PubMed Abstract \| Publisher Full Text\n\nRay WA, Meador KG: Antipsychotics and sudden death: is thioridazine the only bad actor? Br J Psychiatry. 2002; 180: 483–4. 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PubMed Abstract\n\nVines SK, Farrington CP: Within-subject exposure dependency in case-crossover studies. Stat Med. 2001; 20(20): 3039–49. PubMed Abstract \| Publisher Full Text\n\nGreenland S: A unified approach to the analysis of case-distribution (case-only) studies. Stat Med. 1999; 18(1): 1–15. PubMed Abstract\n\nScott LJ, Goa KL: Galantamine: a review of its use in Alzheimer’s disease. Drugs. 2000; 60(5): 1095–122. PubMed Abstract\n\nDaly MP: Diagnosis and management of Alzheimer disease. J Am Board Fam Pract. 1999; 12(5): 375–85. PubMed Abstract \| Publisher Full Text\n\nRaskind MA, Peskind ER, Wessel T, et al.: Galantamine in AD: A 6-month randomized, placebo-controlled trial with a 6-month extension. The Galantamine USA-1 Study Group. Neurology. 2000; 54(22): 2261–8. PubMed Abstract\n\nThacker EL, Schneeweiss S: Initiation of acetylcholinesterase inhibitors and complications of chronic airways disorders in elderly patients. Drug Saf. 2006; 29(11): 1077–85. 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Reference Source" }	[ { "id": "6950", "date": "04 Feb 2015", "name": "Qayyim Said", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall Comments: This study explored methods for evaluating ADRs in claims data using an example of effects of AChEIs treatment on Dementia patients. Overall, the study is very well done. However, there are a few issues that need to be clarified as following:While identifying patients with first incident course of AChEI therapy, did the authors apply any criteria for dosage, strength, or duration of the incident treatment? What was the index date for the treated group? The authors explicitly refer to the index date for the untreated group, but not for the treated group. Why did the authors choose \"death\" as one of the outcomes? Was it based on any prior evidence in the case of AChEI therapy? On page 5, in the last paragraph of left panel, I am not sure what exactly the authors are trying to say in the following sentence: \"Clinical endpoints were intended to measure increased health care utilization associated with specific diagnoses.\" It may be helpful to clarify it. The authors have done well to balance measured confounding by using propensity score methods. However, there may also unmeasured (unobserved) confounding in clinical settings that may have implications for some of the results (e.g. the inability to find some of the ADRs). In future studies, methods to control for unobserved confounding (e.g. instrumental variables) may also be used. While discussing (page 11) reasons for inconsistencies in the results from the two study designs, the authors speculate that their \"inability to find an acute increase of gastrointestinal events in the first-treatment time window may be attributable to insensitivity of claims-based coding to identify symptoms of gastrointestinal disturbance.\" The authors attribute similar reason for failing to find evidence of psychological disturbances. It would be helpful if the authors could provide an example of insensitivity of claims-based coding to identify symptoms in a disease state.", "responses": [ { "c_id": "1282", "date": "01 Apr 2015", "name": "Brian Sauer", "role": "Reader Comment", "response": "Thank you for your kind and thoughtful review of our article. We will answer your questions in the order they were asked:\"While identifying patients with first incident course of AChEI therapy, did the authors apply any criteria for dosage, strength, or duration of the incident treatment?\"No we did not attempt to look at duration of therapy or a dose response relationship. We used a more traditional analysis that attempted to mimic a intention-to-treat design. This is a great suggestion and we may consider applying an on-protocol type analysis that attempts to compare continuous treatment to those who never started treatment. \"What was the index date for the treated group? The authors explicitly refer to the index date for the untreated group, but not for the treated group.\"The index date for the treated was the day the medication was dispensed for a new (incident) course of therapy \"Why did the authors choose \"death\" as one of the outcomes? Was it based on any prior evidence in the case of AChEI therapy?\"Death was not an expected or theoretical adverse outcome. To be consistent we should have simply characterized death as one of the unexpected outcomes evaluated. Nice point. \"On page 5, in the last paragraph of left panel, I am not sure what exactly the authors are trying to say in the following sentence: \"Clinical endpoints were intended to measure increased health care utilization associated with specific diagnoses.\" It may be helpful to clarify it.\"Because expected outcomes were not rare and occurred in much of the population prior to treatment initiation we decided to use a recurrent event analysis. Most pharmacoepidemiological studies try to measure the incident event. We only did this for death. The analysis of recurrent episodes of disease or symptoms compares the difference in \"disease activity or healthcare utilization for these conditions\" instead of incident occurrence of the conditions. \"The authors have done well to balance measured confounding by using propensity score methods. However, there may also unmeasured (unobserved) confounding in clinical settings that may have implications for some of the results (e.g. the inability to find some of the ADRs). In future studies, methods to control for unobserved confounding (e.g. instrumental variables) may also be used.\"This is a nice suggestion and we will consider appropriate instruments and discuss whether should attempt this. \"While discussing (page 11) reasons for inconsistencies in the results from the two study designs, the authors speculate that their \"inability to find an acute increase of gastrointestinal events in the first-treatment time window may be attributable to insensitivity of claims-based coding to identify symptoms of gastrointestinal disturbance.\" The authors attribute similar reason for failing to find evidence of psychological disturbances. It would be helpful if the authors could provide an example of insensitivity of claims-based coding to identify symptoms in a disease state.\"This statement was speculation on our part. The rationale is that prescribers expected GI and psychological disturbance and may have just viewed that as an expected reaction to treatment and not a treated condition and therefore was not coded. Chart review would be required to verify these statements and we didn't have access to the medical notes for this study." } ] }, { "id": "9151", "date": "01 Jul 2015", "name": "Yajaira M. Bastardo", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall Comments: This study presents a framework for discovering ADRs using population based administrative data sources. The approach is exemplified by use of AChEIs treatment on dementia. Overall, the study is very well done. However, there is an issue that needs to be clarified as following:On page 4, paragraph 3, the authors state that they treated the use of statin drugs as indicators of health status because they are preferably prescribed to less frail who are not at the end of life. Even though the statement seems reasonable the reference they provided does not support it.The results of these pilot study shows that use the framework for discovering ADRs using population based administrative data a source is promising as a hypothesis generating approach.", "responses": [] }, { "id": "9448", "date": "10 Jul 2015", "name": "Alison Bourke", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting paper, and as stated, it would be useful to see the methodology repeated on larger datasets to increase the power. One small point - the authors say that their inability to find an acute increase of gastrointestinal events in the first-treatment time window may be attributable to insensitivity of claims-based coding to identify symptoms of gastrointestinal disturbance. Could this also be because they were only looking for the outcome as a primary diagnosis, and GI disturbance may be consider less important? Would it have been possible to use prescribing of certain therapeutic agents as a proxy of GI events", "responses": [ { "c_id": "1458", "date": "13 Jul 2015", "name": "Brian Sauer", "role": "Author Response", "response": "Alison, thank you for reviewing our work. Your are right that the choice to look at primary diagnosis may have made it difficult to detect GI disturbance. There has also been criticism that Medicaid primary diagnosis for outpatient coding doesn't consistently represent the cause of the visit. As a result of this earlier criticism we repeated the analysis using all ICD9 codes and our findings were not qualitatively different. Your idea to use prescribing of therapeutic agents as a proxy for GI events is clever. We may repeat this work in the VA system using current data and we will consider additional measures of GI disturbance. We will also have access to the medical notes to search for key words and validate text based approaches to detecting \"mild to moderate\" side effects that may not get coded.I also should have done a better job of integrating this framework into existing population frameworks, such as OMOP. Much of this work was done before the popularization of concepts like \"negative controls\". We treated death as the \"negative control\" and attempted to identify known effects, explore theoretical adverse effects and screen for common drug related adverse effects that were not expected based on published literature. We didn't expect to find an association with hematological effects. Review of the literature and google sites found a provider observed an increase in hematological adverse effects in his population of patients. A followup study is needed." } ] } ]	1	https://f1000research.com/articles/3-258
https://f1000research.com/articles/3-257/v1	28 Oct 14	{ "type": "Correspondence", "title": "Hot topics at the intersection of aging and energetics: Diabetes/insulin resistance, Sirtuins, and the Microbiome", "authors": [ "Dudley W. Lamming" ], "abstract": "A recent review in F1000Research identified the “top research priorities identified in leading publications” at the interface of Aging and Energetics. The authors identified the ten most-cited papers in each of the years 2010 through 2013, and used these forty papers to identify thematic categories. However, the search methodology used by the authors omitted many high-impact aging manuscripts. Minor modifications in the authors’ search methodology finds that Diabetes/insulin resistance, Sirtuins, and the Microbiome are also top thematic categories.", "keywords": [ "My laboratory members and I were excited to read a recent review in F1000Research1", "entitled “Aging and energetics’ ‘Top 40’ future research opportunities 2010–2013”. The review discusses research opportunities in 10 fields at the intersection of aging and metabolism. The authors identified ten research categories through a Scopus search for the ten most-cited papers in each of the years 2010 through 2013", "for a total of 40 papers. However", "upon examining the list of these papers", "I was surprised to notice that a significant number of these articles were reviews", "and that I recognized very few of the papers. I was also struck by the relatively low number of citations each paper had gathered", "as well as by the almost complete absence of high-impact journals such as Cell", "Nature", "Science and PNAS from the search results." ], "content": "Correspondence\n\nMy laboratory members and I were excited to read a recent review in F1000Research1, entitled “Aging and energetics’ ‘Top 40’ future research opportunities 2010–2013”. The review discusses research opportunities in 10 fields at the intersection of aging and metabolism. The authors identified ten research categories through a Scopus search for the ten most-cited papers in each of the years 2010 through 2013, for a total of 40 papers. However, upon examining the list of these papers, I was surprised to notice that a significant number of these articles were reviews, and that I recognized very few of the papers. I was also struck by the relatively low number of citations each paper had gathered, as well as by the almost complete absence of high-impact journals such as Cell, Nature, Science and PNAS from the search results.\n\nHelpfully, the authors included their Scopus search terms in the methods section of their review, and I was easily able to replicate the results of their search. Upon consideration of the search terminology, I realized the authors had limited their results to papers in which “aging”, “lifespan” and other key search terms relating to aging and metabolism appeared in the title. I repeated their search with one important difference: I searched for keywords and did not limit my search to the title field (see Methods). My list of the 10 most cited articles from each year (2010–2013) at the intersection of aging and metabolism/energetics is given in Appendix A.\n\nThe list I generated is almost entirely different from the authors’ results, including only 3 of the 40 papers identified by Allison and colleagues. My search identified many more high impact papers (based on citation count) and a greater proportion, 18 out of 41 papers, were published by the high impact journals Cell, Nature, Science and PNAS. Whereas Allison et al. identified Morselli et al.2 as the original research manuscript in 2010 with the most citations (99), my methodology identified 8 papers from 2010 with a higher number of citations, the highest of which, Ng et al.3, had 298 citations.\n\nI performed a rapid scoring of my resulting list against the thematic categories identified by Allison and colleagues. Interestingly, “nutrient effects beyond energy” rose to the top of this list, due in part to my inclusion of metformin-related papers in this category. A new thematic area, Diabetes/insulin resistance, was tied for second place with Mitochondria, reactive oxygen species, and cellular energetics. This was followed by another new thematic area, “Sirtuins”, tied with “Calorie restriction”. The Microbiome was also an important new thematic area. Three categories identified by Allison et al., “use and effects of mesenchymal stem cells (MSCs)”, “accretion and effects of body fat”, and “the aging heart”, were entirely absent from my newly generated list.\n\nIn conclusion, a minor change in search methodology resulted in the retrieval of a very different list of papers than that identified by Alison and colleagues. “Calorie restriction”, “nutrient effects beyond energy”, “mTOR”, and “autophagy” are still clearly important areas for aging research. However, Diabetes/insulin resistance, Microbiome, and Sirtuins should be included in this list. The narrow search strategy employed by Allison et al. missed these important thematic areas.\n\n\nMethods\n\nI repeated the Scopus search the authors1 had performed, substituting the use of the “KEY” (keyword) field in place of the “TITLE” (title) field. The search performed was:\n\n( ( KEY ( ( aging OR ageing OR lifespan OR longevity OR senescence ) ) AND PUBYEAR > 2009 AND PUBYEAR < 2014 ) AND ( KEY ( ( calori* OR diet* OR energetic* OR nutri* OR food OR fat OR adipo* OR \"body composition\" ) ) AND PUBYEAR > 2009 AND PUBYEAR < 2014 ) )\n\nFor each search year (2010–2013), I then excluded review articles and removed self-citations using the Scopus tools, and then manually removed additional review articles and articles that did not involve biological research in the field of aging (i.e., consensus treatment guidelines, articles on children in developing countries, and material science manuscripts). Eleven manuscripts were included in 2012 due to a 10th place tie in citation count.\n\nThematic areas were annotated by inspection of the title and abstract only. Themes were taken from the list generated by Allison et al., and the following areas were added after inspection of the titles only: Diabetes/insulin resistance, Microbiome, and Sirtuins. Proteostasis was added to the Autophagy category, and Metformin was added to the “Nutrient effects beyond energy” category during the scoring process.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nI would like to thank D. Cohen for critical reading of this correspondence. I would like to thank all the members of the Lamming lab, especially S.I. Arriola Apelo. This work was supported using facilities and resources from the William S. Middleton Memorial Veterans Hospital. This work does not represent the views of the Department of Veterans Affairs or the United States Government.\n\n\nSupplementary materials\n\nAppendix A: Top ten most cited papers on aging and energetics using keyword field for each year between 2010 and 2013.\n\nAn alternative top 40 list to that generated by Allison et al.4 using the KEY field in Scopus instead of the TITLE field.\n\nClick here to access the data.\n\nhttp://dx.doi.org/10.5256/f1000research.5625.s37562\n\n\nReferences\n\nAllison DB, Antoine LH, Ballinger SW, Bamman MM, Biga P, Darley-Usmar VM, et al.: Aging and energetics' 'Top 40' future research opportunities 2010–2013 [v1; ref status: indexed, http://f1000r.es/4ae]. F1000Res. 2014; 3: 219. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMorselli E, Maiuri MC, Markaki M, Megalou E, Pasparaki A, Palikaras K, et al.: Caloric restriction and resveratrol promote longevity through the Sirtuin-1-dependent induction of autophagy. Cell Death Dis. 2010; 1: e10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNg SF, Lin RC, Laybutt DR, Barres R, Owens JA, Morris MJ: Chronic high-fat diet in fathers programs β-cell dysfunction in female rat offspring. Nature. 2010; 467(7318): 963–6. PubMed Abstract \| Publisher Full Text\n\nAllison DB, Antoine LH, Ballinger SW, Bamman MM, Biga P, Darley-Usmar VM, et al.: Appendix A in: Aging and energetics' 'Top 40' future research opportunities 2010–2013 [v1; ref status: indexed, http://f1000r.es/4ae]. F1000Res. 2014; 3: 219. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "6566", "date": "05 Nov 2014", "name": "David Lombard", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis piece by Dr. Lamming is informative and concise. The search methodology and results are well described. It should be of interest to researchers in biogerontology.", "responses": [] }, { "id": "6563", "date": "24 Nov 2014", "name": "Holly M. Brown-Borg", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe observations noted in this manuscript partially reflect the subjectiveness of this type of analysis. The results of both manuscripts describe key publications in the field that are skewed unintentionally by search terms.", "responses": [] }, { "id": "6807", "date": "25 Nov 2014", "name": "Monica Driscoll", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nLamming comments on, and presents an alternative approach to, identification of “hot topics” in aging research previously published by Allison et al. (2014). In the original paper, Allison et al. screened title to identify the 10 most cited papers from 2010 to 2013 and to suggest promising future research opportunities. Lamming was concerned about including review articles in the search, and sensed a lack of concentration of papers listed in the highest impact factor journal. By broadening the search to include title as well as key words, and excluding review articles, Lamming generated a markedly different list of top cited papers. Interestingly, distinct thematic areas (which make intuitive sense as hot topics) were identified, including diabetes/insulin resistance, sirtuins, and microbiome. Bottom line: changing search approach somewhat modestly had a major impact on list outcome. The correspondence is interesting and the search was validly conducted. The paper makes the important point well familiar to geneticists—design of the screen has a major impact on outcome. Both search strategies have merit, and the reader who prefers not to search for her/himself now has two chances to have her/his papers or research area cited.", "responses": [] } ]	1	https://f1000research.com/articles/3-257
https://f1000research.com/articles/3-131/v1	19 Jun 14	{ "type": "Research Article", "title": "Secretomes of apoptotic mononuclear cells ameliorate neurological damage in rats with focal ischemia", "authors": [ "Patrick Altmann", "Michael Mildner", "Thomas Haider", "Denise Traxler", "Lucian Beer", "Robin Ristl", "Bahar Golabi", "Christian Gabriel", "Fritz Leutmezer", "Hendrik Jan Ankersmit", "Patrick Altmann", "Michael Mildner", "Thomas Haider", "Denise Traxler", "Lucian Beer", "Robin Ristl", "Bahar Golabi", "Christian Gabriel", "Fritz Leutmezer" ], "abstract": "The pursuit of targeting multiple pathways in the ischemic cascade of cerebral stroke is a promising treatment option. We examined the regenerative potential of conditioned medium derived from rat and human apoptotic mononuclear cells (MNC), rMNCapo sec and hMNCapo sec, in experimental stroke.We performed middle cerebral artery occlusion on Wistar rats and administered apoptotic MNC-secretomes intraperitoneally in two experimental settings. Ischemic lesion volumes were determined 48 hours after cerebral ischemia. Neurological evaluations were performed after 6, 24 and 48 hours. Immunoblots were conducted to analyze neuroprotective signal-transduction in human primary glia cells and neurons. Neuronal sprouting assays were performed and neurotrophic factors in both hMNCapo sec and rat plasma were quantified using ELISA.Administration of rat as well as human apoptotic MNC-secretomes significantly reduced ischemic lesion volumes by 36% and 37%, respectively. Neurological examinations revealed improvement after stroke in both treatment groups. Co-incubation of human astrocytes, Schwann cells and neurons with hMNCapo sec resulted in activation of several signaling cascades associated with the regulation of cytoprotective gene products and enhanced neuronal sprouting in vitro. Analysis of neurotrophic factors in hMNCapo sec and rat plasma revealed high levels of brain derived neurotrophic factor (BDNF).Our data indicate that apoptotic MNC-secretomes elicit neuroprotective effects on rats that have undergone ischemic stroke.", "keywords": [ "The search for clinically effective strategies to intercept the deleterious events that follow a stroke is ongoing. This quest has been particularly driven by the limitations that the use of tissue plasminogen activator (tPA) poses in patients with ischemic stroke (Fonarow et al.", "2011). For Europe", "it is projected that stroke events will increase from 20% in 2002 to 35% in 2050 in the population above 65 years of age (Truelsen et al.", "2006). The United States report a yearly incidence of approximately 795", "000", "killing about 135", "000 people each year (Roger et al.", "2012). Most patients have to settle for the need of specialized care culminating into a burden to both persons affected and health care systems (Strong et al.", "2007). Even though the potential of targeting neuroprotective pathways to treat ischemic stroke has been debated extensively", "there seems to be a consensus towards more multilayered strategies (Iadecola & Anrather", "2011)." ], "content": "Introduction\n\nThe search for clinically effective strategies to intercept the deleterious events that follow a stroke is ongoing. This quest has been particularly driven by the limitations that the use of tissue plasminogen activator (tPA) poses in patients with ischemic stroke (Fonarow et al., 2011). For Europe, it is projected that stroke events will increase from 20% in 2002 to 35% in 2050 in the population above 65 years of age (Truelsen et al., 2006). The United States report a yearly incidence of approximately 795,000, killing about 135,000 people each year (Roger et al., 2012). Most patients have to settle for the need of specialized care culminating into a burden to both persons affected and health care systems (Strong et al., 2007). Even though the potential of targeting neuroprotective pathways to treat ischemic stroke has been debated extensively, there seems to be a consensus towards more multilayered strategies (Iadecola & Anrather, 2011).\n\nSeveral neuroprotective proteins that play a role in the ischemic cascade have been identified and studied, such as cAMP response element-binding protein (CREB), Akt, extracellular-signal regulated kinase (Erk1/2), or heat shock protein 27 (HSP27) (Roux & Blenis, 2004), (Qi et al., 2012). The transcription factor CREB, for instance, exerts its neuroprotective role in the ischemic response by activating protective genes and trophic factors such as B-cell lymphoma 2 (Bcl-2) or brain-derived neurotrophic factor (BDNF) (Wilson et al., 1996). The protein-chaperone HSP27 inhibits pathways leading to cell death (van der Weerd et al., 2010). Finding a treatment targeting these proteins simultaneously could open a new window of opportunity in acute stroke management and regeneration.\n\nThe role of different populations of adult stem cells is being investigated in several fields of regenerative medicine. Distant stem cells track sites of injury and counteract tissue damage (Assmus et al., 2011; Brunner et al., 2008; Lagasse et al., 2000). It is hypothesized that human mesenchymal stem cells (hMSCs) produce cytokines and growth factors that subsequently repair damaged tissues, including the brain (Chen et al., 2001). After homing to the injured areas, where hypoxia, apoptosis, and inflammation occur, hMSCs secrete trophic factors that enable endogenous repair (Joyce et al., 2010). Hence, the scientific community endeavored to either infuse or inject stem cells in multiple organ-specific disease entities with only limited clinical success in myocardial infarction (Jensen & Patterson, 2013; Lemmens & Steinberg, 2013; Wollert & Drexler, 2010).\n\nIn 2005, Thum et al. postulated in their “dying stem cell hypothesis” that therapeutic stem cells are already in the state of apoptosis while being processed for treatment, thus causing immune suppression by scaling down the adaptive and innate immune system (Thum et al., 2005). The authors speculated that these “therapeutic apoptotic stem cells” are able to attenuate hypoxia-induced inflammation (Saas et al., 2010), (Fadok et al., 2001).\n\nWe have extended this concept and utilized suspensions of apoptotic peripheral blood mononuclear cells (MNCs), rather than stem cells themselves, as a therapeutic agent for the treatment of rodent myocardial infarction in a previous study (Lichtenauer et al., 2011), (Ankersmit et al., 2009), (Hoetzenecker et al., 2012). The almost complete absence of long term myocardial scarring led us to newly validate the suitability of peripheral MNCs and their secreted factors for regenerative medicine. In our previous work we showed that first, human apoptotic MNC-secretomes (hMNCapo sec) circumvented inflammation and caused preferential homing of c-kit+/CD34- endothelial progenitor cells; second, hMNCapo sec caused immune suppression in vitro and, third, paracrine factors derived from human apoptotic MNC-secretomes led to an upregulation of matrix metalloproteinase 9 (MMP-9) and Interleukin 8 (IL-8) in primary cultured human fibroblasts. Both these factors are known to be involved in neoangiogenesis (Nold-Petry et al., 2010). In addition, hMNCapo sec has been shown to cause enhanced wound healing in vivo via the formation of new blood vessels and increased migration of primary cultured fibroblasts and keratinocytes (Mildner et al., 2013). Ultimately, one single intravenous administration of hMNCapo sec in a large animal closed chest reperfusion model of acute myocardial infarction (AMI) prevented myocardial damage (Lichtenauer et al., 2011), (Ankersmit et al., 2009), (Hoetzenecker et al., 2012).\n\nWith these data at hand, we investigated in the present study whether secretomes derived from rat (rMNCapo sec) and human (hMNCapo sec) apoptotic MNC are also able to reduce ischemic lesion volumes and improve the neurological outcome in a rat MCAO (middle cerebral artery occlusion) model.\n\n\nMaterials and methods\n\nAll animal procedures were approved by the Animal Research Committee of the Medical University of Vienna (Protocol No.: 66.009/127-II/3b/2011) in accordance to the guidelines for the Care and Use of Laboratory Animals by the National Institutes of Health. Efforts were made to minimize suffering.\n\nFor the production of human MNC-secretomes, human MNCs were isolated from whole blood of healthy volunteers. This was approved by the ethics committee of the Medical University of Vienna (approval number: EK 2010/034). Participants provided their written informed consent.\n\nA total of 84 adult male Wistar Rats (Charles River Laboratories, Sulzfeld, Germany) weighing 280–320 g were used. Animals were kept in cages of three to four and accustomed to a 12 hour light-dark cycle for two weeks. Nutrition and tap water were provided ad libitum. Sixteen animals made up the secretome group; out of which MNCs were extracted out of whole blood and their secretomes produced. Ten animals were randomly selected for the pilot-phase with the intention of establishing protocol and attaining a consistent surgical technique. The remaining 58 animals were used for the study groups and randomly assigned to either the control or the treatment group. Deaths occurred equally in both study groups (total N=21, n=10 in setting 1 [rMNCapo sec] and n=11 in setting 2 [hMNCapo sec], 7 animals were excluded from the study (n=3 in setting 1 and n=4 in setting 2) and euthanized within 6 hours after MCAO due to severe dyspnea and suffering. For statistical analyses, the remaining 30 animals were used (n=16 in setting 1 and n=14 in setting 2) Data were analyzed in a blinded manner. Surgery was performed by a surgeon unaware if the animals received treatment or placebo. Neuroscore was evaluated by an investigator not involved in the surgical procedure or application of compounds. Statistical analyses were performed by an external statistician.\n\nThis section describes the production of apoptotic MNC-secretomes derived from rats. These were used for the treatment group in setting 1 whereas the treatment group in setting 2 received apoptotic MNC-secretomes derived from humans (see below). Syngeneic rat-MNCs were harvested from splenocytes of Wistar rats. For this procedure, animals were anesthetized with an intraperitoneal administration of Ketamine (100 mg/kg) and Xylazine (10 mg/kg). Through a midline incision, spleens were harvested and MNCs were separated by passing spleens through 70 µm and 40 µm cell strainers (BD Biosciences, Vienna, Austria). Red blood cells were lyzed for 90 seconds using a red blood cell lysing buffer (Sigma Aldrich, Vienna, Austria). After washing, MNCs were resuspended in 4 mL CellGro serum-free medium (CellGenix GmbH, Freiburg, Germany) and apoptosis was induced by Caesium-137 irradiation (Department of Transfusion Medicine, Vienna General Hospital) with 45 Gy. Cells were cultivated in CellGro serum-free medium at a concentration of 25×106 cells/mL at 37°C and 5% CO2 for 18 hours. Cells were removed by centrifugation at 1300 RPM for 9 minutes (Beckman Coulter Allegra® X-15R, Brea, CA, USA) and cell culture supernatants were then dialyzed against 50 mM ammonium acetate (Sigma Aldrich) using dialysis membranes (cut off: 6–8 kDa; Spectrum laboratories, Breda, The Netherlands) for 24 hours at 4°C on a shaking platter. Subsequently, the dialyzed supernatants were lyophilized over night (Lyophilizator Christ alpha 1–4, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). Lyophilization was performed at −20°C and 0,1 mbar pressure. The final product, rMNCapo sec, was stored at -80°C. For the control group, the same cell culture medium that was used for the production of rMNCapo sec was irradiated, cultivated, dialyzed, and lyophilized accordingly. All cell and tissue samples were handled under sterile conditions. Microbial smears on chocolate agars (BD Biosciences, Vienna, Austria), a non-selective media for cultivation of fastidious microorganisms, were performed before lyophilization to rule out contaminations.\n\nPathogen-reduction methods such as photodynamic treatment with methylene blue (MB) plus visible light and gamma radiation have been developed to inactivate viruses and other pathogens in plasma and platelet concentrates. Regulatory authorities require these two pathogen reduction steps for blood derived products such as IVIg, plasma or coagulation factors (Gauvin & Nims, 2010; Lambrecht et al., 1991; Nims et al., 2011; Wallis & Melnick, 1965) to be performed in a good manufacturing practice (GMP) facility.\n\nHuman apoptotic MNC-secretomes were prepared as described previously (Lichtenauer et al., 2011). Briefly, venous blood samples (75 mL) were drawn from healthy volunteers (n=15). Blood cells were separated using Ficoll-Paque (GE Healthcare Bio-Sciences AB, Stockholm, Sweden) density gradient centrifugation. MNCs were resuspended with CellGro serum-free medium. After irradiation with 45 Gy as described above, cells were cultivated with CellGro serum-free medium at a concentration of 25×106 cells/mL under sterile conditions for 18 hours. Supernatants were collected by centrifugation and methylene blue (MB) plus light treatment was performed in the Theraflex MB-Plasma system (MacoPharma, Mouvaux, France) using the Theraflex MB-Plasma bag system (REF SDV 0001XQ) and an LED-based illumination device (MacoTronic B2, Maco- Pharma). Light energy was monitored and reached 120 J/cm2. Integrated in the bag system was a pill containing 85 mg of MB, yielding a concentration range of 0,8 to 1,2 mM MB per unit. Removal of MB and photoproducts by Blueflex filtration was done consecutively after treatment. Air was removed from the plasma units before illumination. After lyophilization of this viral inactivated cell culture supernatant as indicated above, the lyophilized powder ran through the second step of pathogen removal, gamma irradiation. Gamma irradiation was performed by Mediscan, which operates a gamma irradiation unit (Gammatron 1500, Mediscan, Seibersdorf, Austria). Gamma rays were generated by radioactive decay of Cobalt 60. For this purpose, apoptotic MNC-secretomes were put in metal sterilization totes that pass on a meandering path through the irradiation vault around the emitting center in five layers. The cobalt unit emits photons that are almost isotropic. Regarding the complex path (280 positions in five layers) the dose is distributed consistently. The dose rate recorded by a Polymethyl methacrylate (PMMA) dosimeter was determined to be 25.000 Gy after 23 hours of irradiation. The lyophilized and two-step pathogen-free supernatant of apoptotic MNCs (hMNCapo sec) was stored at -80°C. For the control group, cell culture medium was put through the same steps (cultivation, two step pathogen reduction, irradiation and lyophilization).\n\nFifty eight animals were weighed and anesthetized with an intraperitoneal administration of Ketamine (100 mg/kg) and Xylazine (10 mg/kg). This was followed by a subcutaneous injection of Piritramide (15 mg/kg). Animals were then intubated with an 18G intravenous catheter (BD Biosciences) and anesthesia was maintained throughout surgery with 1,5% isoflurane delivered in 1,5 L air and 0,8 L oxygen per minute. Body temperature was regulated using a heating pad (Trixie 76085 Heizmatte, Trixie, Flensburg, Germany). Permanent middle cerebral artery occlusion (MCAO) of the right hemisphere was performed according to the suture model described by Zea Longa et al. using a coated monofilament (Doccol Corporation, CA, USA) (Longa et al., 1989). Briefly, a 3 cm coated monofilament with a thickened tip was inserted into the external carotid artery (ECA) and advanced to the middle cerebral artery (MCA) to induce ischemia in the MCA territory.\n\nIn the first experimental setting, lyophilized rMNCapo sec (produced from 12.5×106 apoptotic rat MNCs) or control medium was resuspended in 0,3 mL saline (Fresenius Kabi, Vienna, Austria) in the laboratory prior to surgery. Forty minutes after MCAO induction, both the treatment group and the control group (n=29) randomly received 0,3 mL rMNCapo sec or control medium intraperitoneally (Figure 1, blue arrow). In the second setting, lyophilized hMNCapo sec (produced from 12.5×106 apoptotic human MNCs) or lyophilized control medium were each resuspended in 0,3 mL saline in the laboratory prior to surgery. In order to investigate whether a higher dosage and time interval would provide additional benefits, animals from experimental setting 2 (n=29) received two intraperitoneal doses 40 minutes and 24 hours after MCAO induction (Figure 1, red arrows). The rationale for this two-step-approach is given in the discussion. In addition, all animals were given a subcutaneous injection of 3,5 mL/kg saline after surgery and put under a heating lamp until they woke up.\n\nFor setting 1, rMNCapo sec (apoptotic MNC-secretomes from rats) were injected 40 minutes after MCAO (blue arrow). In setting 2, hMNCapo sec (apoptotic MNC-secretomes from humans) were administered twice at 40 minutes (0,7 hours) and 24 hours after MCAO (red arrows). In both settings, neurological evaluations were performed at 0 hours (before surgery) as well as 6, 24, and 48 hours after surgery (boxes). Both treatment and control animals were euthanized 48 hours after surgery and brain slices were treated with TTC (2,3,5-triphenyltetrazolium chloride) to stain ischemic areas in the brain.\n\nIn both experimental settings, animals were euthanized 48 hours after surgery with an intraperitoneal injection of 600 mg/kg Pentobarbital. Brains were harvested and cut into five 2 mm coronal slices using a brain matrix (Zivic Instruments, Pittsburgh, PA, USA) and razor blades (Zivic Instruments). In order to stain ischemic areas, brain slices were then incubated for 30 minutes at 37°C in a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC; CarlRoth, Karlsruhe, Germany) (Bederson et al., 1986). Slices were digitalized using a commercially available photo scanner (Epson Perfection V330 Scanner; Figure 2). Lesion volumes were determined by a blinded investigator using ImageJ planimetry software (Version 1.6.0_10; Rasband, W.S., ImageJ, U.S. National Institutes of Health; Bethesda, MD, USA). Lesion volumes were calculated with respect to edema formation using the following formula: 100×(Volume of the contralateral hemisphere-Volume of the ipsilateral hemisphere)/(Volume of the contralateral hemisphere). Ipsi- and contralateral lesion volumes were calculated by multiplication of area with slice thickness summed for all sections (Swanson et al., 1990).\n\nBrains were stained with a 2% solution of TTC forty-eight hours after MCAO. Animals received either treatment (in this representative scan: hMNCapo sec) or control medium, in this case, 40 minutes 24 hours after surgery. White areas indicate ischemic tissue while red areas stain for non-ischemic tissue. Animals treated with control medium (left image) had larger ischemic (= white) areas than animals treated with hMNCapo sec (right image).\n\nNeurological examinations were performed by a blinded investigator in both experimental settings using a neurological score before surgery, and 6, 24, and 48 hours after surgery. The test at each time point consists of seven exercises and animals would receive a score ranging from 0 points (no pathological responses) to 7 points (maximum impairment). A successfully completed exercise would add 0 points to the score. Pathological performance in an exercise would add 1 point. The exercises were: left forepaw extension, instability to lateral push from right, tail hanging, walking on ground, whisker movement on the left, hearing, and vision (Nedelmann et al., 2007). E.g. if the rats could not hold against a hand pushing them from the right, they would get 1 point. If they did push against the examiner’s hand, they would get 0 points. Accordingly, if the rats did extend their left forepaw when hauled up by their tail, they would get 0 points. If they could not extend their left forepaw, they would get 1 point. This adds up to a score ranging from 0 points (= no pathological response at all), to 7 points (= highly impaired animal).\n\nHuman primary astrocytes, Schwann cells and neurons were obtained from CellSystems (CellSystems Biotechnologie, St. Katharinen, Germany) and cultured in their respective growth medium (CellSystems) at 37°C and 5% CO2.\n\n3×106 astrocytes, Schwann cells and 3×105 neurons were seeded in 6-well plates (Costar, Vienna, Austria) and cultured overnight in their respective growth medium. After removal of the medium, cells were washed twice at room temperature with PBS (Gibco BRL, Gaithersburg, MA, USA) and cultured in their respective basal medium (astrocyte or Schwann cell growth medium (Cell Systems) without growth supplements) for 3 hours. Aliquots of lyophilized human MNC-secretome and control medium were resolved in the different basal media at a 10-fold concentration (lyophilized secretome, derived from 25×106 cells/mL). One tenth of this solution was then directly added to the cell cultures. After 1 hour, the cells were washed at room temperature with PBS and lyzed in 200 µl SDS-PAGE loading buffer (100 ml contain: 1g SDS (Sigma, Vienna, Austria), 3 mg EDTA (Sigma) and 0.75g TRIS (Sigma). pH is adjusted with HCl (Merck, Vienna, Austria) to 6,8) for 10 minutes at room temperature. After sonication (Laborpartner, Vienna, Austria: output = 100%; 20 Cycle for 1 second each) and centrifugation (20000g for 10 minutes) proteins were size-fractionated by SDS-PAGE through an 8 to 18% gradient gel (Amersham Pharmacia Biotech, Uppsala, Sweden) and transferred to nitrocellulose membranes (BioRad, Hercules, CA, USA). Immunodetection was performed with anti-c-Jun (Cell Signaling Technology, Inc. Danvers, MA, USA; 1µg/ml, #9165), anti-phospho-c-Jun (Cell Signaling Technology; 1µg/ml, #9261), anti-CREB (Cell Signaling Technology; 1µg/ml, #9197), anti-phospho-CREB (Cell Signaling Technology; 1µg/ml, #9198), anti-Akt (Cell Signaling Technology; 1µg/ml, #2938), anti-phospho-AKT (Cell Signaling Technology; 1µg/ml, #9271), anti-Erk1/2 (Cell Signaling Technology; 1µg/ml, #4695), anti-phospho-Erk1/2 (Cell Signaling Technology; 1µg/ml, #4376), anti-HSP27 (Cell Signaling Technology; 1µg/ml, #2402), anti-phospho-Hsp27 (Cell Signaling Technology; 1µg/ml, #2404) followed by an HRP-conjugated goat anti-mouse IgG antiserum or a goat anti-rabbit IgG antiserum (GE Healthcare, Freiburg, Germany). Reaction products were detected by chemiluminescence with the ChemiGlow reagent (Biozyme Laboratories Limited, South Wales, U.K.) according to the manufacturer’s instructions.\n\nTo investigate neuronal sprouting of human primary neurons, 1×104 cells (CellSystems) were seeded in 24-well plates (Costar) and allowed to adhere for 24 hours. Cells were further cultivated in neuronal medium (see above) without growth factors for five days together with the secretome of hMNC derived from 2,5×106 cells/mL (hMNCapo sec) or control medium. After five days cells were fixed at room temperature in 100% methanol for 10 minutes and stained with methylene blue (Sigma; 0.5% in methanol). Excess methylene blue was washed out with distilled water, and culture wells were evaluated with an inverted microscope (EvosXL, Life Technologies, Carlsbad, CA, USA). Cell cultures were digitalized using an Olympus Digital Camera E-520 (3648×2736 pixels). For measurement of neurite lengths, a representative area (1420×2456 pixels) of the photographed cell cultures was determined and set by a blinded observer using Adobe Photoshop Lightroom Software (Version 5.2, 2013; Adobe, San José, CA, USA). Neurite lengths within this specific area were measured by a blinded investigator using ImageJ software (Bethesda, MD).\n\nBDNF, nerve growth factor (NGF) and glial derived neurotrophic factor (GDNF) in rMNCapo sec, hMNCapo sec, and control medium, were measured using commercially available ELISA-kits (Enzyme linked immunosorbent assay; BDNF: catalog# DY248; beta-NGF catalog# DY256; GDNF catalog# DY212; R&D Systems, Minneapolis, MN, USA). All samples were assayed in triplicates. Manufacturer’s instructions were followed and plates were read at 450 nm on a Wallac Multilabel counter 1420 (PerkinElmer, Boston, MA, USA).\n\nTo see whether intraperitoneal administration of apoptotic MNC-secretomes and control medium influence BDNF production, six rats were injected intraperitoneally with hMNCapo sec (n=3), or control medium, (n=3). Rats were euthanized with an intraperitoneal injection of 600 mg/kg Pentobarbital 24 hours after injection with hMNCapo sec or control medium. Blood was retrieved in heparinized tubes, centrifuged, and plasma was stored at -20°C. Rat BDNF levels were measured using a commercially available BDNF-ELISA (catalog# KA0330, Abnova, Taipei, Taiwan) and plates were read at 450 nm on a Wallac Multilabel counter 1420 (PerkinElmer, Boston, MA, USA).\n\nTo test for differences in lesion volume between control and treatment groups the Mann-Whitney U-test was applied. Differences were also assessed graphically using box-plots. The calculations were performed separately for each experimental setting. Linear mixed models were calculated to explain neuroscores by time point, treatment and interaction effects between time point and treatment. A random intercept term was included for each individual animal to account for the correlation of observations within an individual. The calculations were done using the MIXED procedure in SAS 9.3. The neuroscores were also assessed graphically by plotting a time curve of mean neuroscore values ± SEM for each group. Neurite lengths in neuronal cultures were compared applying the student‘s t-test. A p-value of 0.05 or below was considered significant.\n\n\nResults\n\nIn a rat model of MCAO we examined the potential of apoptotic MNC-secretomes to reduce ischemic lesion volumes in an allogeneic (experimental setting 1, rMNCapo sec) and a xenogeneic approach (experimental setting 2, hMNCapo sec) (Figure 1). The results of the allogeneic setting displayed significantly lower lesion volumes in the treatment group compared to the control group as shown by TTC-staining (Figure 3). Treatment with rMNCapo sec led to a mean decrease of 36% in total infarct volume. Hemispheric lesion volumes (mean±SD) in the control group were 59%±8% ranging from 50% to 73% (Mann Whitney U-test; p=0,0006; Figure 3a). The treatment group had a mean hemispheric lesion volume of 38%±11% ranging from 24% to 51%. In the xenogeneic setting, we injected hMNCapo sec 40 minutes and 24 hours after MCAO. The reduction of the infarction volume in the xenogeneic setting was statistically significant and comparable to that observed in the allogenic setting (Mann Whitney U-test; p=0,0041; Figure 3b). The mean decrease in total infarct volume was 37%. Hemispheric lesion volumes (mean±SD) in the control group were 52%±8% ranging from 42% to 67%. The treatment group had a mean hemispheric lesion volume of 33%±11% ranging from 21% to 48%.\n\nThe percentage of hemispheric lesion volumes (%HLV) are represented as box and whiskers plots, wherein the boxes indicate the 1st and the 2nd quartile and the whiskers the minimum and maximum within 1.5 times the interquartile range from the box. Figure 3a shows lesion volumes as the extend of ischemia in setting 1, where apoptotic MNC-secretomes derived from rats were administered 40 minutes after MCAO compared to controls. Figure 3b corresponds to setting 2, where apoptotic MNC-secretomes derived from humans were administered 40 minutes and 24 hours after MCAO. In both settings, MNCapo sec (red boxes) caused a significant decrease in infarct volumes (p=0,0006 for setting 1, Figure 3a, and p=0,0041 for setting 2, Figure 3b) compared to the control group (white boxes) that received only cell culture medium.\n\nIn order to discover the effects of apoptotic MNC-secretomes on the neurological outcome, we performed a neurological exam on each animal at 4 specific time points. The first score was measured prior to surgery (baseline; 0 hours) and was 0 points for all animals. This was followed by 3 postoperative measurements at 6, 24 and 48 hours. All animals expressed neurological stroke-symptoms immediately after anesthesia wore off. The fixed effect coefficient estimates of the mixed models, their standard errors and p-values are shown in Tables 1a and 1b. The factor Treatment was coded in a way that Treatment=0 corresponds to the control group and Treatment=1 corresponds to the treatment group. The time point 6 h is the reference group for the factor Time.\n\nData include both experimental setting 1 that used apoptotic MNC-secretomes derived from rats, “rMNCapo sec”, (Table 1a), and setting 2 that used apoptotic MNC-secretomes derived from humans, “hMNCapo sec”, (Table 1b). p-values are calculated from t-tests with the null hypothesis of the true coefficient being equal to 0. The factor Treatment was coded in a way that Treatment=0 corresponds to the control group and Treatment=1 corresponds to the treatment group. The time point 6 h is the reference group for the factor Time.\n\nThe results from both settings are similar. At time point 6 h there is no significant mean difference in the neuroscores between control and treatment, which is suggested by the coefficient for Treatment. The coefficients for Time 24 h and Time 48 h are not significantly different from zero. We can therefore not conclude that the mean neuroscore in the control group changes with time. The interaction terms for Treatment and Time, however, are significant. This indicates a significant decrease in the mean neuroscore over time in the treatment group. These results correspond well to the graphical depictions of the time curves (Figures 4a and 4b).\n\nMean neuroscores (±SEM) are plotted over time. Treated animals (red triangles for setting 1, Figure 4a, and red squares for setting 2, Figure 4b) improved over time compared to controls (black/white triangles for setting 1, Figure 4a, and black/white squares for setting 2, Figure 4b). Error bars correspond to +/- one standard error.\n\nGlia cells are non-neuronal cells that provide support and protection for neurons in the brain and peripheral nervous system (Giaume et al., 2010). We therefore investigated the potency of hMNCapo sec to activate/phosphorylate signaling-molecules that are part of protective pathways in human primary astrocytes as well as in Schwann cells. For Western blot analysis, astrocytes and Schwann cells were treated with hMNCapo sec for 1 hour. Both cell types showed an increased phosphorylation of CREB, Erk1/2, c-Jun, and Akt. Phosphorylation of HSP27 was only detected in astrocytes (Figure 5).\n\nCell extracts were prepared after stimulation with hMNCapo sec or cell culture Medium as control. Western blot analysis revealed an activation of CREB, ERK1/2, c-Jun, Akt, and HSP27. Proteins were normalized to the respective non-phosphorylated proteins.\n\nWe next investigated whether hMNCapo sec are also effective in human primary neuron cultures. Western blot analysis of neurons and astrocytes revealed a rapid dose dependent activation of CREB phosphorylation (Figure 6a). Incubation of neurons with apoptotic MNC-secretomes led to a significant increase in the length of newly sprouting neurons. Cultured neurons treated with hMNCapo sec had a mean neurite length of 21,04±1,1642 µm (mean±SEM) versus 12,56±1,119 µm in neurons treated with control medium (t-test; *p<0,0001; Figure 6b–c).\n\n(a) Cell extracts were prepared after stimulation with hMNCapo sec or cell culture medium as control. Western blot analysis for phospho-CREB revealed a dose dependent activation of CREB in astrocytes and in neurons. (b) Neuron cultures treated with hMNCapo sec or control medium for five days were stained with methylene-blue. One representative picture of ten is shown. Bar = 10µm (c) Lengths of neurons treated with hMNCapo sec or cell culture medium as control were calculated using ImageJ software. Bars represent the mean of five different cultures.\n\nIn order to characterize the composition of neurotrophic factors present in hMNCapo sec, we performed ELISA for BDNF, GDNF and NGF. Interestingly, only high amounts of BDNF (356,6±13,66 pg/mL) were detected in hMNCapo sec, suggesting an exclusive role for this neurotropic factor in hMNCapo sec (Figure 7).\n\nSix animals received an intraperitoneal injection with hMNCapo sec (n=3, red bar) or control medium (n=3, black/white bar) and BDNF plasma levels were determined 24 hours after administration using ELISA. Results are given as mean±SEM.\n\nAfter revealing BDNF as one component in hMNCapo sec, BDNF levels in plasma of rats treated with hMNCapo sec were measured with ELISA 24 hours after i.p. administration. Plasma BDNF-levels were higher in rats treated with hMNCapo sec than in control medium (n=3 for each compound). Twenty four hours after treatment with hMNCapo sec, BDNF plasma levels were 72,78±5,048 ng/mL (mean±SEM) compared to undetectably low BDNF levels in controls (Figure 7).\n\n\nDiscussion\n\nFor over a decade, MSCs have been known to have beneficial effects on the outcome of several disease entities (Assmus et al., 2011; Chen et al., 2001; Crigler et al., 2006; Lagasse et al., 2000). In our previous studies we were able to show that apoptotic MNC-secretomes share some of the regenerative characteristics of stem cells and, based on the data presented in this work, considerably more. We show here that apoptotic MNC-secretomes derived from both rats (rMNCapo sec) and humans (hMNCapo sec) caused a reduction of lesion volumes in rats subjected to MCAO. Neurological evaluations revealed an improvement in motor and sensory function, which was not observed in the control group. Furthermore, apoptotic MNC-secretomes derived from humans (hMNCapo sec) (i) activate several mechanisms ultimately leading to the expression of protective proteins in cultured primary human glial cells, such as astrocytes, Schwann cells and human neurons, and (ii) induce notable sprouting of neurites in primary neuron cultures. Additionally, hMNCapo sec contain BDNF and lead to increased presence of BDNF in plasma of rats treated with hMNCapo sec.\n\nCrigler and coworkers described the ability of MSCs to express neuro-regulatory molecules and to promote neuronal cell survival (Crigler et al., 2006). In the literature, MSCs are described to home to injured areas and regenerate damaged tissue by either causing cytoprotection, anti-inflammation or by inducing activation of endogenous stem cells (Gnecchi et al., 2012; Siegel et al., 2012; Williams & Hare, 2011). Recently, it became commonly accepted that possible stem cell effects are derived from the paracrine factors secreted by MSCs (Di Santo et al., 2009; Jayaraman et al., 2013). This theory of paracrine factors aiding in regenerative processes emerges as a possible explanation for the therapeutic potential of apoptotic MNC-secretomes shown previously in myocardial infarction and, given our new data, in ischemic stroke (Ankersmit et al., 2009; Lichtenauer et al., 2011a; Lichtenauer et al., 2011b). The mechanisms of action seem to be a matter of immunomodulation and cytoprotection (Ankersmit et al., 2009; Lichtenauer et al., 2011a; Lichtenauer et al., 2011b). Cultured glial cells incubated with apoptotic MNC-secretomes revealed an upregulation of several proteins involved in conveying cytoprotective signals, such as CREB, HSP27, Erk 1/2, and Akt. These results suggest that apoptotic MNC-secretomes affect different pathways within the ischemic cascade, and most prominently they appear to act via anti-apoptotic pathways. Since glial cells are known to support and protect neurons, it is tempting to speculate that the enhanced cell survival of glial cells is also beneficial for neurons via indirect mechanisms. However, our data on CREB phosphorylation and neurite sprouting also suggest direct positive effects of apoptotic MNC-secretomes on neurons. The effects followed by activation of these signaling molecules are indeed protective. The activation of the transcription factor CREB induces BDNF, which plays an important role in neuronal protection (Ferrer et al., 2001). In addition, the overexpression of HSP27 resulted in a 30% reduction of infarct sizes in rats subjected to MCAO (van der Weerd et al., 2010). The extracellular signal-regulated kinases Erk 1/2, part of the MAPK-families (mitogen activated protein kinase), are thought to play a role in cell survival and proliferation (Roux & Blenis, 2004). Furthermore, activation of the prosurvival kinase Akt reduces the proapoptotic signaling that is triggered by ischemia. It is suggested that Akt activation protects against ischemic brain injury by suppressing the proapoptotic JNK3 (c-Jun N-terminal kinase-3) pathway (Zhang et al., 2007). Studying the brain’s ischemic cascade, where repair processes are initiated through the expression of several of these aforementioned survival proteins, an upregulation of protective proteins and pathways by a targeted treatment is obviously a welcome effect (Moskowitz, 2010).\n\nWith regard to further mechanisms of neuronal regeneration and protection, we show here that apoptotic MNC-secretomes contain BDNF and enhance systemic, most likely indirect, BDNF secretion in rats after injection with human apoptotic MNC-secretomes. It was shown that neurotrophic factors may play a critical role in the treatment of cerebral ischemia (Abe, 2000). It was discussed in the literature that BDNF is among the most important neurotrophic factors due to its capability to promote neurogenesis and angiogenesis, to prevent neuronal cell death, and to modulate local inflammatory processes (Jiang et al., 2011; Ploughman et al., 2009; Schäbitz et al., 2007). In light of these data it seems arguable that apoptotic MNC-secretomes provide, at least in part, indirect protection in this experimental stroke model via BDNF. When we integrate all these recent findings into our suggested mode of action of apoptotic MNC-secretomes in ischemic stroke, we can, step by step, characterize it as a biological, battling the ischemic cascade on several fronts.\n\nWe decided to run two experimental settings in order to investigate (i) whether syngeneic rMNCapo sec are able to attenuate ischemic lesion volumes and (ii) to further define whether xenogenic hMNCapo sec, produced according to GMP criteria and virus-inactivated, are equally potent as rMNCapo sec. This extended two-step experimental approach was chosen because hMNCapo sec are very close to the final product that is intended for later clinical use. hMNCapo sec can be produced effectively and securely out of whole blood, similar to blood products such as packed red cells. Also, allogeneic apoptotic MNC-secretomes derived from multiple healthy donors could be pooled and lyophilized. In the future, the proposed ability of hMNCapo sec to reduce ischemic injury in the early phases of stroke may even imply its use in combination with established therapeutic concepts such as arterial recanalization. Considering that inflammatory processes are particularly aggressive in ischemia associated with reperfusion, the suppression of inflammation provided by hMNCapo sec would be a fitting addition to reperfusion therapy (Stowe et al., 2009). Experiments investigating the anti-inflammatory action of apoptotic MNC-secretomes in our setting of experimental MCAO are currently being performed.\n\n\nConclusion\n\nWe suggest that apoptotic MNC-secretomes have multifaceted, direct and indirect, neuroprotective characteristics acting through different rays in the ischemic cascade (inflammation, apopotosis, ischemia). Rats treated with apoptotic MNC-secretomes in this experimental stroke study expressed smaller lesion volumes than control animals and showed improvement in neurological function over time. Based on our findings, we believe that apoptotic MNC-secretomes derived from human blood can aid in the development of new treatment strategies in ischemic stroke.\n\n\nData availability\n\nfigshare: Apoptotic MNC-secretomes in experimental stroke. doi: 10.6084/m9.figshare.1051645 (Altmann et al., 2014).", "appendix": "Author contributions\n\n\n\nStudy design: PA, HJA, TH, CG\n\nIn vivo studies: PA, TH, DT\n\nIn vitro studies: MM, PA, DT, LB, BG\n\nStatistical analyses: RR\n\nManuscript: PA, HJA, FL\n\n\nCompeting interests\n\n\n\nThe Medical University of Vienna has claimed financial interest (Patent number: PCT/EP09/67534, filed 18 Dec 2008; Patent name: Pharmaceutical preparation comprising supernatant of blood mononuclear cell). Hendrik Jan Ankersmit is a shareholder of APOSIENCE AG, which owns the rights to commercialize apoptotic MNC-secretomes for therapeutic use. All other authors declare that they have no competing interests. APOSIENCE AG is a funder of his study.\n\n\nGrant information\n\nThis study was funded by the Christian Doppler Laboratory for cardiac and thoracic diagnosis and regeneration.\n\n\nReferences\n\nAbe K: Therapeutic potential of neurotrophic factors and neural stem cells against ischemic brain injury. J Cereb Blood Flow Metab. 2000; 20(10): 1393–1408. PubMed Abstract \| Publisher Full Text\n\nAnkersmit HJ, Hoetzenecker K, Dietl W, et al.: Irradiated cultured apoptotic peripheral blood mononuclear cells regenerate infarcted myocardium. Eur J Clin Invest. 2009; 39(6): 445–456. PubMed Abstract \| Publisher Full Text\n\nAssmus B, Iwasaki M, Schächinger V, et al.: Acute myocardial infarction activates progenitor cells and increases Wnt signalling in the bone marrow. 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Data Source" }	[ { "id": "5480", "date": "21 Jul 2014", "name": "Johannes Boltze", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article by Altmann and colleagues assesses the therapeutic impact of conditioned medium from both human and rodent apoptotic mononuclear cells (MNC). The paper reports highly interesting findings that not only contribute significantly to the understanding of adult cell-based therapies for ischemic diseases, but also shade some different light on the field. Of particular value is the inclusion of a tailored statistical model by a professional statistician as this enhances reliability of results and their interpretation. Moreover, the method section is extensive, allowing easy reproduction of experiments by other labs. In summary, the article is clearly above the average quality of similar studies, highly informative, and relevant. There are, however, a number of minor weak points that may be corrected or at least require some more detailed discussion.Please report all available physiological parameters of animals during MCAO and details on the randomization strategy applied. Why was the treatment started in a time window of <1h after stroke onset. This is clinically irrelevant and prevents collection of post-stroke baseline function. What was the rationale for applying secretomes from rat cells just one time, but two times from human cells? Why was the injection done i.p. and not i.v. or i.a. as common in the field? Behavioral data should be presented as mean +/- standard deviation. For details, please see Carter (2013). Please explain the rational of the surprisingly short post-stroke surveillance period of just 48 hours. Both STEPS and STAIR committees recommend at least 1 month of post-stroke observation in preclinical studies, so there should be strong argument for underscoring this significantly. The discussion relies heavily on MSC which, potentially, is a bit distracting. There are ample papers discussing mechanisms of therapeutic impact by MNC, primarily from umbilical cord and bone marrow. The discussion should be directed towards those populations. There are ample commas (as typical in German) in decimal numbers that should be replaced by periods.", "responses": [ { "c_id": "1031", "date": "15 Oct 2014", "name": "Patrick Altmann", "role": "Author Response", "response": "Thank you for your valuable and kind comments. Having included them in our manuscript, we feel that we were able to further increase the value of our manuscript.Please report all available physiological parameters of animals during MCAO and details on the randomization strategy applied.We agree that physiological parameters during surgery ensure the well-being of our animals, however after careful consideration we chose to avoid additional strain on our animals (through cannulizations of blood vessels, etc.). Preparing and designing our study, we found that other studies also abstained from invasive monitoring during surgery1 2 3. To our knowledge, the model we used represents a well-established and often published procedure and therefore posesses predictable characteristics.Regarding the randomization process, the surgeon was blinded to treatment. After surgery, the animals‘ tails were labeled and observers blinded to treatment and previous scores assessed neurological function. We clarified this in the manuscript (Materials and Methods). Why was the treatment started in a time window of <1h after stroke onset. This is clinically irrelevant and prevents collection of post-stroke baseline function. What was the rationale for applying secretomes from rat cells just one time, but two times from human cells? Why was the injection done i.p. and not i.v. or i.a. as common in the field?Thank you for mentioning this. We are aware of the time \"issue“ and realize that our setting might be partially out of the ordinary. Since we already had the most experience with apoptotic MNC-secretomes administered 40 minutes after myocardial infarction we decided to start our stroke research at this time window as well. This first investigation was mostly intended to show the potential of apoptotic MNC-secretomes in stroke rather than setting a particular time window for our treatment or being able to tell the best dosage for its application. We are aware of the limitations of this study design and we agree that additional experiments need to be done in order to see how the administration of apoptotic MNC-secretomes at different or, for that matter, later time points affect outcome after ischemic stroke.We applied the “human” dose twice in order to see if there was an additional benefit for the doubled dose. We agree, though, that rat and human apoptotic MNC-secretomes are hardly comparable, representing a limitation of this study. To avoid possible loss of potency in the xenogenic setting (human MNC-secretomes in rat MCAO) we decided to use treatment twice compared to only once in the allogeneic experiments (rat MNC-secretomes in rat MCAO). This approach further allowed us to reduce the required amount of animals according to the principle of the three R’s (replacement, refuction and refinement) 4.Concerning the injection route, we chose to administer our compounds intraperitoneally because (i) we felt it was easier to do, (ii) we had the most experience with applying medication via i.p. injections, and (iii) we considered it less invasive than intravenous application. Also, when we designed this study, we found some articles that did describe use of i.p. injections 5 6 7. Behavioral data should be presented as mean +/- standard deviation. Thank you for letting us know about this. We corrected this as requested. Please explain the rational of the surprisingly short post-stroke surveillance period of just 48 hours. Both STEPS and STAIR committees recommend at least 1 month of post-stroke observation in preclinical studies, so there should be strong argument for underscoring this significantly.We absolutely agree that our small study does not meet STEPS/STAIR criteria. This very first investigation with all its agreeable limitations was done to see if the promising data obtained from previous studies in other fields of regenerative medicine might be translatable into experimental ischemic stroke. We realize that larger animal studies with a more thorough observation and surveillance in accordance with STAIR criteria are needed to further uncover effects of human apopototic MNC-secretomes in experimental stroke. We added this information to the Discussion (second paragraph). The discussion relies heavily on MSC which, potentially, is a bit distracting. There are ample papers discussing mechanisms of therapeutic impact by MNC, primarily from umbilical cord and bone marrow. The discussion should be directed towards those populations.Thank you for showing this confusion. The first paragraph of the Discussion is intended to summarize what we know about MSCs and how we came to use secretomes of MNCs in our previous research and how we learned that they actually share some of the regenerative characteristics of MSCs. The second paragraph should point out how actually paracrine factors rather than stem cells themselves account for their regenerative properties and how this was observed in several studies. After that we discuss that our data indicates that neurotrophic factors involved in protective pathways seem to be triggering that therapeutic effect that we saw in our experiments. This is also mentioned in the Discussion and we included a new reference 8. There are ample commas (as typical in German) in decimal numbers that should be replaced by periods.Thank you. We replaced all ample commas in decimal numbers with periods. References 1. Zhou L, Li F, Xu HB, Luo CX, et al.: Treatment of cerebral ischemia by disrupting ischemia-induced interaction of nNOS with PSD-95.Nat Med.2010; 16 (12): 1439-1443 PubMed Abstract \| Publisher Full Text 2. Son HY, Han HS, Jung HW, Park YK: Panax notoginseng Attenuates the Infarct Volume in Rat Ischemic Brain and the Inflammatory Response of Microglia.J Pharmacol Sci.2009; 109 (3): 368-379 PubMed Abstract \| Publisher Full Text 3. Elango C, Devaraj SN: Immunomodulatory effect of Hawthorn extract in an experimental stroke model.J Neuroinflammation.2010; 7 (97). PubMed Abstract \| Free Full Text \| Publisher Full Text 4. Smith R: Animal research: the need for a middle ground.BMJ. 2001; 322 (7281): 248-249 PubMed Abstract \| Free Full Text \| Publisher Full Text 5. Ahmad AS, Yun YT, Ahmad M, Maruyama T, et al.: Selective blockade of PGE2 EP1 receptor protects brain against experimental ischemia and excitotoxicity, and hippocampal slice cultures against oxygen-glucose deprivation.Neurotox Res.2008; 14 (4): 343-351 PubMed Abstract6. Sahin S, Alkan T, Temel SG, Tureyen K, et al.: Effects of citicoline used alone and in combination with mild hypothermia on apoptosis induced by focal cerebral ischemia in rats.J Clin Neurosci.2010; 17 (2): 227-231 PubMed Abstract \| Publisher Full Text 7. Barakat W, Safwet N, El-Maraghy NN, Zakaria MN: Candesartan and glycyrrhizin ameliorate ischemic brain damage through downregulation of the TLR signaling cascade.Eur J Pharmacol.2014; 724: 43-50 PubMed Abstract \| Publisher Full Text 8. Korf-Klingebiel M, Kempf T, Sauer T, Brinkmann E, et al.: Bone marrow cells are a rich source of growth factors and cytokines: implications for cell therapy trials after myocardial infarction.Eur Heart J.2008; 29 (23): 2851-2858 PubMed Abstract \| Publisher Full Text \| Reference Source" } ] }, { "id": "5568", "date": "25 Jul 2014", "name": "Michael Chopp", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors in this report examined the regenerative potential of conditioned medium derived from rat and human apoptotic mononuclear cells (MNC) in experimental stroke. They found that administration of rat as well as human apoptotic MNC-secretomes (rMNCapo sec and hMNCapo sec) significantly reduced ischemic lesion volumes and improved neurological function after stroke in both treatment groups. Furthermore, co-incubation of human astrocytes, Schwann cells and neurons with hMNC-secretomes resulted in activation of several signaling cascades associated with the regulation of cytoprotective gene products and enhanced neuronal sprouting in vitro. These data indicate that apoptotic MNC-secretomes elicit neuroprotective effects on rats that have undergone ischemic stroke. Overall, this is an interesting study. However; there are some important pieces of information missing that should be addressed to improve the manuscript: The authors should include the references for their protocol on inducing apoptotic rat MNCs, as well as the references for the protocol on rat MNC preparation. Data need be presented showing apoptosis and characterizing of their rat MNCs. The authors mentioned in the Materials and Methods that they will discuss the rationale for the two-step-approach of their hMNCapo sec administration. However, this rationale is absent from the text. It is important to address why the authors employed two dosages instead of one dose (like the rMNCapo sec). In the part “Neuronal sprouting assay”, the authors should describe with more precision the methods they employed for measurement of neurite length. In the part “Determination of BDNF in rat plasma”, the authors should include the dose and amount of hMNCapo sec they administered to rats. In the Results “Apoptotic MNC-secretomes induce CREB phosphorylation and neuronal sprouting in human primary neurons and contain BDNF”, the authors state that “Interestingly, only high amounts of BDNF (356,6±13,66 pg/mL)were detected in hMNCapo sec, suggesting an exclusive role for this neurotrophic factor in hMNCapo sec (Figure 7).” But these data are not shown in Figure 7. The authors used a comma symbol as decimal point, and the number of significant figures should be uniform throughout the manuscript. Typos in the text, e.g. “rays” in third line of the conclusion part should be “ways”.", "responses": [ { "c_id": "1032", "date": "15 Oct 2014", "name": "Patrick Altmann", "role": "Author Response", "response": "Thank you for your helpful report. Your suggestions have allowed us to make our manuscript better and present our data more accurately. The authors should include the references for their protocol on inducing apoptotic rat MNCs, as well as the references for the protocol on rat MNC preparation. Data need be presented showing apoptosis and characterizing of their rat MNCs.Thank you for this suggestion. We conducted an additional experiment which allowed us to compare apoptosis rates of irradiated and non-irradiated MNCs using Annexin/PI staining and flow cytometry (please see “new” Figure 1). For our active compound, we found that 85% of rat MNCs that had been irradiated and cultured for 18 hours were apoptotic.Also, we referenced the piece of work where we generated and utilized rat apoptotic MNC‑secretomes for the first time. The authors mentioned in the Materials and Methods that they will discuss the rationale for the two-step-approach of their hMNCapo sec administration. However, this rationale is absent from the text. It is important to address why the authors employed two dosages instead of one dose (like the rMNCapo sec).Thank you for letting us know that this explanation has gotten lost somehow. Since we already had the most experience with apoptotic MNC-secretomes administered 40 minutes after myocardial infarction we decided to start our stroke research at this time window as well. This first investigation was mostly intended to show the potential of apoptotic MNC-secretomes in stroke rather than setting a particular time window for our treatment or being able to tell the best dosage for its application. We are aware of the limitations of this study design and we agree that additional experiments need to be done in order to see how the administration of apoptotic MNC-secretomes at different or, for that matter, later time points affect outcome after ischemic stroke.We applied the “human” dose twice in order to see if there was an additional benefit for the doubled dose. We agree, though, that rat and human apoptotic MNC-secretomes are hardly comparable, representing a limitation of this study. To avoid possible loss of potency in the xenogenic setting (human MNC-secretomes in rat MCAO) we decided to use treatment twice compared to only once in the allogeneic experiments (rat MNC-secretomes in rat MCAO), This approach further allowed us to reduce the required amount of animals according to the principle of the three R’s (replacement, reduction and refinement)1. In the part “Neuronal sprouting assay”, the authors should describe with more precision the methods they employed for measurement of neurite length.Thank you for your comment. A blinded observer set a random area of the photographed cell culture (as seen in Figure 7b) using Adobe Photoshop software (i) of cells treated with human apoptotic MNC-secretomes and (ii) of cells treated with cell culture medium (control). The areas in each photograph measured 1420x2456 pixels. Subsequently, they picked and marked visible distinct, full-length and non-overlapping 30-35 neurites using ImageJ software. Another blinded investigator measured the lengths of these marked neurites using ImageJ software. We added this more detailed explanation to the Materials and Methods section. In the part “Determination of BDNF in rat plasma”, the authors should include the dose and amount of hMNCapo sec they administered to rats.Thank you for making us aware of this. We added this to the manuscript. As in all studies here, we used secretomes of 12.5 million apoptotic MNCs. In the Results “Apoptotic MNC-secretomes induce CREB phosphorylation and neuronal sprouting in human primary neurons and contain BDNF”, the authors state that “Interestingly, only high amounts of BDNF (356,6±13,66 pg/mL)were detected in hMNCapo sec, suggesting an exclusive role for this neurotrophic factor in hMNCapo sec (Figure 7).” But these data are not shown in Figure 7.Thank you for showing this confusion. You can see that we now added a new panel to this Figure (now “new” Figure 8) with panel (a) showing how we only traced BDNF in our compounds and panel (b) showing BDNF levels of rats treated with our compounds. The authors used a comma symbol as decimal point, and the number of significant figures should be uniform throughout the manuscript.Thank you for mentioning this. We replaced all commas by decimal points throughout the manuscript. Also, the number of significant figures are now uniform throughout the manuscript in order to increase readability. Typos in the text, e.g. “rays” in third line of the conclusion part should be “ways”.Thank you. We proof-read the manuscript a couple more times. References 1. Smith R: Animal research: the need for a middle ground.BMJ. 2001; 322 (7281): 248-249 PubMed Abstract \| Free Full Text \| Publisher Full Text" } ] }, { "id": "5562", "date": "13 Aug 2014", "name": "An Zhou", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this work, the authors examined the therapeutic potential of secretory proteomes of apoptotic mononuclear cells (MNCapo sec) in treating focal ischemic stroke, modeled on rats with permanent MCAO. They found that intraperitoneal administration of MNCapo sec prepared from either rat splenocytes or human circulating blood could significantly reduce MCAO-induce brain infarct. The authors also attempted to identify protein factors in the MNCapo sec that may play a role in the observed neuroprotection, and suggested BDNF. Further, in an in vitro setting, the authors analyzed changes in levels of a number of proteins known to mediate anti-apoptotic responses or neuronal regeneration in three different types of cell cultures, namely astrocytes, Schwann cells and neurons, upon treating the cultures with MNCapo sec. An increase in some of selected proteins led to the suggestion of activation of several cytoprotective singnaling cascades. The topic of the work is of significance in developing feasible therapeutics for stroke. The same group has previously published on the use of MNCapo sec in treating myocardial infarction. Thus the current findings are supportive of the potential of MNCapo sec. Concerns exist, however, on several issues: Lack of description of irradiation-treated MNC, especially in the splenocyte-derived cultures. Though referred to as apoptotic MNC, no experimental data were shown to demonstrate the induction and the extent of irradiation-induced apoptosis, for the period during which the conditioned media were collected. Do the two cell cultures (rat splenocytes-derived and human PBMC-derived) respond to irradiation with comparable characteristics? Are their secretomes comparable? Please provide evidence on whether proteins in the intraperitoneally administered MNCapo sec may have reached the brain region of MCAO territory. This information would be instrumental in helping to understand the mechanisms that underlie the action of MNCapo sec. Post-MCAO neurological examinations were conducted at 6, 24 and 48 hours. The covered period was short in regard to the development of infarction and neurological impairments. For the MNCapo sec, please provide protein quantities administered. Different cell cultures or the same culture under different conditions, even with identical cell numbers, may differ in secretion rates. The rationale for using three different cultures in vitro to investigate the cellular response upon MNCapo sec application is somewhat unconvincing. Astrocytes, Schwann cells and neurons may react to MNCapo sec administration differently in brain in vivo. Why not perform immunohistochemical analyses of proteins of interests on brain sections? The determination of plasma BDNF levels included only 3 animals per data point. Was there a power determination to justify this small sample size? Wording: in the title, the abstract and manuscript text, there appeared a somewhat interchangeable use of “ameliorate neurological damage”, “regenerative potential” and “promote neuronal cell survival”. Please revise to be more accurate and concise.", "responses": [] } ]	1	https://f1000research.com/articles/3-131
https://f1000research.com/articles/3-143/v1	01 Jul 14	{ "type": "Software Tool Article", "title": "Cytoscape tools for the web age: D3.js and Cytoscape.js exporters", "authors": [ "Keiichiro Ono", "Barry Demchak", "Trey Ideker", "Barry Demchak", "Trey Ideker" ], "abstract": "In this paper we present new data export modules for Cytoscape 3 that can generate network files for Cytoscape.js and D3.js. Cytoscape.js exporter is implemented as a core feature of Cytoscape 3, and D3.js exporter is available as a Cytoscape 3 app. These modules enable users to seamlessly export network and table data sets generated in Cytoscape to popular JavaScript library readable formats. In addition, we implemented template web applications for browser-based interactive network visualization that can be used as basis for complex data visualization applications for bioinformatics research. Example web applications created with these tools demonstrate how Cytoscape works in modern data visualization workflows built with traditional desktop tools and emerging web-based technologies. This interactivity enables researchers more flexibility than with static images, thereby greatly improving the quality of insights researchers can gain from them.", "keywords": [ "Cytoscape was born as a GUI-based", "Java desktop application in 20031", "2. Today", "it is a de-facto standard application for biological network analysis and visualization. Around 2005", "Java was one of the dominant programming languages for data visualization applications", "and Java-based feature-rich toolkits were developed3. However", "since they were designed before the re-discovery of Ajax4", "developers could not predict the success of JavaScript and related web technologies today. Cytoscape is still an important platform for biological network data integration and analysis", "but for data visualization and sharing", "we need a new method to take advantage of modern web technologies. Utilizing HTML5 and other emerging web technologies", "Cytoscape Consortium developed a JavaScript library for network visualization called cytoscape.js (http://cytoscape.github.io/cytoscape.js/)", "the successor of Cytoscape Web5", "to meet the demand from the Cytoscape user community. Although Cytoscape and Cytoscape.js share some of the core concepts", "such as Visual Styles or automatic layouts", "they are completely independent software packages and there has been no simple way to use Cytoscape data sets in Cytoscape.js." ], "content": "Introduction\n\nCytoscape was born as a GUI-based, Java desktop application in 20031,2. Today, it is a de-facto standard application for biological network analysis and visualization. Around 2005, Java was one of the dominant programming languages for data visualization applications, and Java-based feature-rich toolkits were developed3. However, since they were designed before the re-discovery of Ajax4, developers could not predict the success of JavaScript and related web technologies today. Cytoscape is still an important platform for biological network data integration and analysis, but for data visualization and sharing, we need a new method to take advantage of modern web technologies. Utilizing HTML5 and other emerging web technologies, Cytoscape Consortium developed a JavaScript library for network visualization called cytoscape.js (http://cytoscape.github.io/cytoscape.js/), the successor of Cytoscape Web5, to meet the demand from the Cytoscape user community. Although Cytoscape and Cytoscape.js share some of the core concepts, such as Visual Styles or automatic layouts, they are completely independent software packages and there has been no simple way to use Cytoscape data sets in Cytoscape.js.\n\nPublic biological data repositories are still growing rapidly and the demand for visualizing those complex biological data sets is high. Traditionally, analysis and visualization of biological data is done by desktop applications, and in most cases, visualizations created by popular libraries (matplotlib6, ggplot27) are static images. It is hard to perform exploratory data analysis only with static images, especially when visualizing large data sets, because some of the details are lost due to the limited size of printed papers or computer screens. Instead of developing custom visualization toolkits for specific data sets, the scientific data visualization community is heading towards web-based technologies to utilize actively developed visualization toolkits such as mpld3 (http://mpld3.github.io/) or Bokeh (http://bokeh.pydata.org/). D3.js8 is one of the most popular toolkits for creating custom interactive visualizations. If biologists can use the existing powerful desktop application and these emerging web technologies for data visualizations and sharing, it opens up a new way to understand large and complex biological data sets.\n\nTo bridge the gap between the desktop version of Cytoscape and other web-based data visualization toolkits, we developed Cytoscape modules to generate web-friendly data formats. The goal is to enable shared visualization of Cytoscape networks via a web platform (e.g., browser), and our strategy is to enable conversion from Cytoscape data objects to a format friendly to web apps, and to provide a template code for creating an interactive web application. In this paper, we present the implementation of Cytoscape data exporters and template web applications and demonstrate how users can publish their data sets as interactive data visualizations with our new tools.\n\n\nImplementation\n\nThe exporter modules were developed for Cytoscape 3. The Cytoscape.js exporter is part of Cytoscape 3 core distribution and is available as a standard feature. The D3.js exporter is an app, and this means developers can write any type of additional JSON exporters as necessary. Exporter modules generate JavaScript Object Notation (JSON) files that are readable by Cytoscape.js and D3.js. To visualize these JSON files as interactive network diagrams, users have to write some JavaScript and HTML5 code to read, map, and render the network and table data in the files. The general structure of code for basic network visualization is common to most use cases. To simplify this visualization task, we implemented template HTML5 projects to render the exported JSON files.\n\nCytoscape.js is a JavaScript library for interactive network visualization developed by Cytoscape Consortium. Although Cytoscape and Cytoscape.js share core concepts, they have completely independent code bases written in Java and JavaScript. This means there is no binary-level compatibility between these two software packages. The purpose of this exporter is to provide data-level compatibility between these software packages. There are two core functions in this exporter module: network/table converter and Visual Style to CSS converter.\n\nConversion from Cytoscape networks and tables to Cytoscape.js JSON is done by a serialization module implemented with Jackson, a Java-based JSON parser library (https://github.com/FasterXML/jackson). The converter takes a Cytoscape network object and associated node, edge, and network tables as inputs and converts them into a single JavaScript object represented as JSON. Most of the basic data types are converted into JSON except the following: nested networks, custom graphics, and node shapes and edge line types, which are only available in Cytoscape.\n\nIn contrast, converting Cytoscape Visual Style is a nontrivial process. A Visual Style in Cytoscape is a collection of visual mapping functions, which is a mapping from data to visual variables9, and default values. Conversion from default visual property values to JavaScript objects is a simple one-to-one mapping with several non-compatible value filterings. In Cytoscape, there are three types of visual mapping functions. They are Passthrough, Continuous, and Discrete. Cytoscape.js has the concept of a visual mapping function in its design and it follows standards of CSS and selectors, which is significantly different from the design of Cytoscape visual mapping functions. The converter translates Cytoscape visual mapping functions into combinations of Cytoscape.js selectors and mappers (Figure 1). This translation absorbs differences in design between the two applications and reproduces Cytoscape Visual Styles as JavaScript objects used for styling in Cytoscape.js.\n\nIn general, D3.js does not have any specific data format for visualization. Generic CSV/TSV tables can be used for all types of visualizations, and its core provides data loaders for those files. An exception is the graph data format for force-layout, which is the basic preset for visualizing graph data in D3.js (https://github.com/mbostock/d3/wiki/Force-Layout). It uses an ordinal (i.e., zero-based) index of nodes as the unique identifier, and edges are represented as a pair of those indices. The D3.js exporter converts Cytoscape network topology into this force-layout format, and transforms all associated data tables into properties of nodes and edges in the JSON.\n\nA tree data structure is a special kind of graph, and D3.js has various types of preset visualizations for it, such as radial layout, circle packing or Treemap. Cytoscape can visualize trees as node-link diagrams, and if we can export tree data models stored as Cytoscape graph objects into a D3.js compatible format, users can create multiple views for same data sets using different visualization techniques which could provide new insights for them. To utilize these presets, the exporter generates tree-style JSON for D3.js. The root node of the tree should be specified manually by the user, then the exporter automatically generates tree JSON with all associated tables.\n\nOnce exporters generate JSON files, users need actual web applications to visualize the data. Both Cytoscape.js and D3.js are designed for developers, not for end-users, and developers are expected to write their own custom visualization code to see the data. Although they are optimized for custom web-based visualizations, basic components of visualization code, including data loading, mapping, and rendering, are common to most applications. To minimize duplicate efforts to visualize the results from JSON exporters, we developed template web application projects to visualize JSON files generated by Cytoscape. These templates can create basic visualizations of the JSON files out of the box. To develop these templates, we used standard tools for modern JavaScript development: Node.js (http://nodejs.org/) as runtime for all development tools, Yeoman (http://yeoman.io/) for code scaffolding, and Grunt (http://gruntjs.com) as task runner.\n\n\nResults\n\nA typical data visualization workflow with our new tools consists of the following four steps. First, users load networks, annotations, and experimental data sets into Cytoscape. Second, utilizing core Cytoscape visualization features, users create custom Visual Styles and layouts. Third, all data sets are exported as JSON files, and finally they can create custom web-based, interactive visualizations from the template projects (Figure 2). The original network data visualized in Figure 3 was imported with a Cytoscape app called KEG-GScape (http://apps.cytoscape.org/apps/keggscape). The advantage of this workflow is that users can use the large collection of existing Cytoscape apps for data integration and analysis, and the result can be exported as interactive web-based visualizations with the new exporters. Figure 3 shows the TCA Cycle pathway which was generated from a KEGG XML (KGML) file, and its Visual Style was automatically generated from the graphics data in the file. Cytoscape.js exporter can generate web-compatible style and network files directly from the Cytoscape view. Our template code for Cytoscape.js is a simple viewer, and it can be used as a basis for complex data visualization application.\n\nThe combination of Cytoscape 3 and new JSON exporters can be used as data integration tool for web-based visualizations. Our sample Grunt project generates a simple template code for visualizing D3.js and Cytoscape.js JSON files.\n\nOriginal network data was imported to Cytoscape 3 using the KEGGScape app. The template code contains minimal set of features like simple table browser and network viewer. The table browser (bottom) is implemented with AngularJS (https://angularjs.org) and Bootstrap (http://getbootstrap.com).\n\nThe exporters create JSON files with both network topology and data tables, and users can create complex data visualizations which cannot be achieved with Cytoscape alone. Figure 4 and Figure 5 shows simple network visualizations created with D3.js force-directed layout (Figure 4) and tree layout (Figure 5). These figures are created with a minimal set of features available in D3.js and they can be used as a “boilerplate” code for custom visualizations. The desktop version of Cytoscape is optimized for rendering node-link, or ball-stick network diagrams, which is only one way to visualize graph data. Cytoscape 3 supports multiple-rendering engines and if developers can implement new rendering engines for new visualizations, such as Treemap or Chord Diagram, they can add new visualizations on Cytoscape. This is not a trivial task and as an alternative, our D3.js templates can be used to make prototypes for new visualizations.\n\n\nConclusions\n\nIn this paper, we presented a new workflow to visualize biological data sets using Cytoscape and modern web-based data visualization libraries. The example visualizations show how users can leverage easy-to-use Cytoscape core features as a part of web-based interactive data visualization publishing workflow.\n\nAt this point, new features discussed in this paper are designed for developers who can write JavaScript and HTML5 code. End users are also a part of our target audience, and so we will implement the “Export as HTML5 Session” feature as a core Cytoscape feature, which creates a compressed archive file that includes all of the networks, tables, and Visual Styles as JSON along with all JavaScript files to visualize the data as a single-page application.\n\n\nSoftware availability\n\nSoftware available from: http://apps.cytoscape.org/apps/d3jsexporter\n\nLatest source code: https://github.com/keiono/cytoscape-d3\n\nSource code as at the time of publication: https://github.com/F1000Research/cytoscape-d3/releases/tag/V1.0\n\nArchived source code as at the time of publication: http://dx.doi.org/10.5281/zenodo.1054710\n\nLicense MIT License\n\nTemplate code generator website https://github.com/cytoscape/cyjs-sample\n\nHigh-resolution images and interactive examples are available at our web sites above.", "appendix": "Author contributions\n\n\n\nKO designed and implemented all software packages and wrote this article. BD worked as a project manager and reviewed this article. TI provided funding and supervision.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was funded by the National Resource for Network Biology (NRNB) under award numbers P41 RR031228 and GM103504 assigned to Trey Ideker.\n\n\nAcknowledgements\n\nWe would like to thank Max Franz and Dr. Gary Bader for their work on the Cytoscape.js JavaScript library.\n\n\nReferences\n\nSmoot ME, Ono K, Ruscheinski J, et al.: Cytoscape 2.8: new features for data integration and network visualization. Bioinformatics. 2011; 27(3): 431–432. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShannon P, Markiel A, Ozier O, et al.: Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003; 13(11): 2498–2504. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeer J, Card SK, Landay JA: prefuse: a toolkit for interactive information visualization. In CHI ’05: Proceedings of the SIGCHI Conference on Human Factors in Computing Systems, page 421, New York, USA, April 2005. ACM Request Permissions 2005; 421–430. Publisher Full Text\n\nGarrett JJ: Ajax: A new approach to web applications. 2005. Reference Source\n\nLopes CT, Franz M, Kazi F, et al.: Cytoscape Web: an interactive web-based network browser. Bioinformatics. 2010; 26(18): 2347–2348. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHunter JD: Matplotlib: A 2D Graphics Environment. Comput Sci Eng. 2007; 9(3): 90–95. Publisher Full Text\n\nWickham H: ggplot2: elegant graphics for data analysis. 2009. Publisher Full Text\n\nBostock M, Ogievetsky V, Heer J: D3: Data-Driven Documents. IEEE Trans Vis Comput Graph. 2011; 17(12): 2301–2309. PubMed Abstract \| Publisher Full Text\n\nBertin J: Semiology of Graphics: Diagrams, Networks, Maps. 1983. Publisher Full Text\n\nOno K, Demchak B, Ideker T: F1000Research/cytoscape-d3. ZENODO. 2014. Data Source" }	[ { "id": "5313", "date": "01 Aug 2014", "name": "Alexander Lex", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article describes an important new development for the Cytoscape project: bridging the gap between complex yet powerful desktop applications to easy-to-use, installation free web application focused on the analysis and presentation. I consider this workflow as an important bridge between the two worlds. The article is technically sound and mostly well written. Some structural comments and concrete suggestions for improving the manuscript are listed below. What I am currently missing in the manuscript is a description of a workflow for cleaning up the exported visualizations. Especially in the KEGG example, the result is rather messy and clearly requires some additional authoring. How would one go about this process? How long would it take? I would suggest adding another figure that shows a post-cleanup version and a paragraph commenting on the process. Also, it would be great to see a comparison of the original layout in Cytoscape in addition to the web-based layouts, ideally for all the results figures, so that readers can clearly judge the quality of the approach. This could also put my previous comment into context, as maybe the KEGG layout isn't much better in Cytoscape standalone, since the KEGG annotations typically don't contain sufficient information about the layout.Structural Suggestions:For this argument: \"However, since they were designed before the re-discovery of Ajax, developers could not predict the success of JavaScript and related web technologies today.\"I would suggest to add that it was simply not feasible at the time since the technology and bandwidth was not available. Regarding this sentence:\"It is hard to perform exploratory data analysis only with static images, especially when visualizing large data sets, because some of the details are lost due to the limited size of printed papers or computer screens.\"I would suggest to also argue that this static approach is especially problematic for networks, as the size of all but trivial networks requires dynamic features to be useful. Here: \"If biologists can use the existing powerful desktop application and these emerging web technologies for data visualizations and sharing, it opens up a new way to understand large and complex biological data sets.\"I think the main point is that it has the potential to combine the best of two worlds: powerful authoring tools and easily shareable interactive results, but I'm not sure whether this is \"a new way to understand[ing]\". Rather it is a new way of engaging users, through a much simplified process. \"via a web platform (e.g., browser)\" -> what would be other web platforms? Or do you mean \"i.e., in the browser\" The hierarchy in the Cytoscape.js exporter section isn't clear. The following two captions seem to be of the same level as the root section. I suggest removing the captions completely and simply make the converters bold in the text. Also, these sections sometimes lack clarity. For example, it is not clear what a Cytoscape Table and Network is. I assume that is clear when one knows the software well, but it would be good to clarify this here for readers who are not that familiar with Cytoscape. What are \"non-compatible value filterings\"? \"The root node of the tree should be specified manually by the user\" -> should or must? Caption Figure 4: \"Visualization of a sample network (galFiltered.sif) by D3.js force-layout.\" -> What is the data shown here? Should a reader know that from the file name? The file name seems irrelevant here. I would rather add a brief description of the data. Some grammar and style suggestions:\"to popular JavaScript library readable formats\" -> to \"formats readable by popular JavaScript libraries.\". \"and Java-based feature-rich toolkits were developed\" -> \"and many Java-based feature-rich toolkits were developed\" . \"technologies, Cytoscape Consortium\" -> \"technologies, the Cytoscape Consortium\". \"libraries (matplotlib, ggplot2)\" -> \"libraries (e.g., matplotlib, ggplot2)\". \"The goal is to enable shared visualization of Cytoscape networks via a web platform (e.g., browser), and our strategy is to enable conversion from Cytoscape data objects to a format friendly to web apps, and to provide a template code for creating an interactive web application.\" -> split into multiple sentences. \"is part of Cytoscape 3 core distribution\" -> \"is part of the Cytoscape 3 core distribution\". \"The D3.js exporter is an app, and this means\" -> \"The D3.js exporter is an app which means\" \"The D3.js exporter is an app, and this means\" -> I suggest clarifying \"app\". Is this a cytoscape plugin? \"we implemented template HTML5 projects to render the exported JSON files.\" -> \"we provide template HTML5 projects to render the exported JSON files.\". \"developed by Cytoscape Consortium\" -> \"developed by the Cytoscape Consortium\". \"There are two core functions in this exporter module: network/table converter and Visual Style to CSS converter.\" -> I would suggest rephrasing this to explain on a high level what these modules do. \"except the following: nested networks,\" -> \"except for nested networks,\". \"converting Cytoscape Visual Style\" -> is this a noun? or do you mean \"Cytoscape's visual styles\". In Figure 1, the code in the \"Continuous Mapping as collection of Mappers\" is unreadable as it is too small. \"Once exporters generate JSON files, users need actual web applications to visualize the data.\" -> \"To visualize the exported JSON files, actual web applications are needed to visualize the data.\". \"visualization code to see the data.\" -> \"visualization code.\" \"Third, all data sets are exported as JSON files, and finally they can create custom web-based, interactive visualizations from the template projects (Figure 2).\" -> This sentence mixes active and passive. I suggest re-writing it in the active voice since the previous sentence is active as well. \"The combination of Cytoscape 3 and new JSON exporters can be used as data integration tool for web-based visualizations\" -> \"The combination of Cytoscape 3 and the new JSON exporters can be used as a data integration tool for web-based visualizations.\". \"created with D3.js force-directed layout\" -> \"created with the D3.js force-directed layout\". \"such as Treemap or Chord Diagram\" -> \"such as Treemaps or Chord Diagrams\". \"License MIT License\" -> \"License: MIT License\". \"Template code generator website: https://github.com/cytoscape/cyjs-sample\" -> \"Template code generator website https://github.com/cytoscape/cyjs-sample\".", "responses": [ { "c_id": "932", "date": "06 Aug 2014", "name": "Keiichiro Ono", "role": "Author Response", "response": "I completely agree with your point about the missing details for web based KEGG visualization part. I will add more details how to reproduce the visualization with this tool. It will be available as a GitHub Wiki page. The following details will be added to the new version:Side-by-side comparison of original Cytoscape visualization and exported versionOriginal Cytoscape Session file and exported JSON files as supplemental dataLink to the instruction wiki page how to reproduce the KEGG visualizationIn addition to these changes, I will fix the structural/grammatical issues in the revised version.Thank you for your helpful suggestions." }, { "c_id": "1095", "date": "25 Nov 2014", "name": "Keiichiro Ono", "role": "Author Response", "response": "Dear Dr. Lex,This version 2 includes all updates for your comments for the original version. Also, recently we released new version of Cytoscape (3.2.0), and it should be compatible with this protocol. If you have problems to test this workflow in the latest environment, please let me know.Best,Keiichiro Ono" } ] }, { "id": "6061", "date": "11 Sep 2014", "name": "Jose Villaveces", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article describes Cytoscape.js and D3.js exporters and a code template generator. Additionally, it provides all the essential information about Cytoscape.js exporter and D3.js exporters and points interested users to the source code for its implementation.Even though there are a few points that require clarification, the tools are a nice addition to Cytoscape and can be seen as a first step in uniting present day web technologies and the more conventional desktop based visualisation provided by Cytoscape.ImprovementsThere is no documentation for the template generator, a README.md file with a few examples will be nice. The D3 example listed in the d3jsexporter page is very small (300 x 150) and the 'View Source' button is not working.", "responses": [] } ]	1	https://f1000research.com/articles/3-143
https://f1000research.com/articles/3-250/v1	24 Oct 14	{ "type": "Research Note", "title": "Assessing the prevalence of malnutrition in tribal children using MUAC as a screening tool", "authors": [ "Mohtashim A. Qureshi", "Ihtesham A. Qureshi", "Asghar Syed", "Suresh Babu Kokku", "Mohtashim A. Qureshi", "Ihtesham A. Qureshi", "Asghar Syed" ], "abstract": "Children malnutrition is a major public health problem in India. Malnutrition has the maximum impact on children living in rural and tribal areas. Various anthropometric indices such as weight–for-age, height-for-age, weight-for- height and Body Mass Index (BMI) are used to assess the nutritional status of the children. Mid-upper-arm circumference (MUAC) is being used as an alternative to traditional measurements like height and weight, particularly in emergency settings. The World Health Organization (WHO) has recommended MUAC to be used as an independent diagnostic criterion for assessing severe acute malnutrition among children. A total of 4502 children between 6-59 months of age were screened over a period of 12 months, in seven Medicins Sans Frontiers (MSF) Project mobile clinic sites located in states of Andhra Pradesh and Chhattisgarh border areas in India. MUAC was measured with MSF-designed fiber optic measuring tapes. In general, the overall prevalence of malnutrition among 6-59 months children was 15.2%. However the prevalence of malnutrition was higher among children of 6-23 months age group (25.8%) as compared to children of 24-59 months (5.4%). Despite various national nutritional intervention programs have been in operation for about four decades, the malnutrition remains very high particularly among the children living in hilly and remote tribal villages.", "keywords": [ "malnutrition", "tribal children", "MUAC", "public health" ], "content": "Introduction\n\nPeriodic growth monitoring of children is an important indicator of the health and nutritional well being of the population. Child undernutrition remains a major public health problem in many countries, and continues to hamper children’s physical growth and mental development1. India registered an impressive growth in term of GDP during last few years, but the malnutrition rates among the Indian children remains high. As reported by UNICEF, in India, about 46% of children below three years have stunting (height-for-age <Median-2SD), while 47% have underweight and 16% are wasted2.\n\nTraditionally, nutritional status was evaluated using anthropometric measures like height, weight and indices like body mass index (BMI)3. However mid-upper-arm circumference (MUAC) is being used as an alternative index of nutritional status for children during famines or refugee crises and as an additional screening tool in non-emergencies, and is based on a single cut-off value for all the children less than five years of age4. Studies showed that under conditions of reduced food intake, lower levels of subcutaneous fat and muscle mass in human arms tend to correspond to a decrease in the MUAC5. In 2005, the World Health Organization (WHO) recommended a MUAC cut-off of 110 mm as an independent diagnostic criterion for severe acute malnutrition. However a higher cut off point of 115 mm was recommended later by WHO as it allows to identify a more accurate number of infants and children with severe acute malnutrition and has a high specificity of more than 99% over the age range 6–60 months6. There is large body of evidence strongly suggesting that MUAC is a better indicator of acute malnutrition than weight/height particularly for use in emergency feeding programs7.\n\n\nAbout MSF, India\n\nSince October 2006, MSF (Médecins Sans Frontièrs, Doctors without Borders) is committed to providing health services to the people in the Naxal-affected regions of Dantewada (Chhattisgarh state) and Khammam (Andhra Pradesh state) in India. MSF India provides impartial medical assistance to the populations with little or no access to health care in these regions. The agency provides primary and secondary healthcare including reproductive health, immunization, health education and treatment of tuberculosis (TB), malaria and diarrhoea among other diseases in conflict-affected areas. MSF runs a Mother and Child Health Centre (MCHC) in Bijapur, Chhattisgarh, also in addition to other mobile clinics that provide health care directly to people in both states8.\n\n\nMaterials and methods\n\nMSF teams carried out MUAC screenings at the Maita, Mallampeta, DharmanaPeta, Pusuguppa, Tippapuram, Yampuram and Puttapalli mobile clinics. MUAC was measured using MSF-designed fiber optic color-coded measuring tapes divided into 2 mm additions12. A girth of the child’s arm within the green part of the tape indicates a normal nutritional status. The yellow part of the tape indicates that the child is at risk of malnutrition, the orange color indicates that the child is moderately malnourished and the red color indicates a severe malnutrition and threat of death [MSF Refugee Handbook] (Table 1).\n\nFrom January 2012 to December 2012, in the above mentioned clinics (Table 2), 2162 children between 6 and 23 months of age and 2340 children between 24 and 59 months of age were screened, making a total of 4502 children. The data were collected over a period of 12 months from seven MSF project clinics in the states of Andhra Pradesh and Chattisgarh namely Maita, Mallampeta, DharmanaPeta, Pusuguppa, Tippapuram, Yampuram and Puttapalli. These mobile clinics are in hard to reach remote hilly tribal villages with poor infrastructural facilities. In addition to MUAC screening, all children attending the mobile clinics with or without health problems were also screened for estimated age which was determined by noting the birth date recorded on the child’s vaccination card. We have limitation on the availability of data for yellow and green colour measurements.\n\n\nResults\n\nAmong the children between 6 and 23 months of age the severe malnutrition (indicated by the red colour) was 3.8%, whereas in children between 24 and 59 months of age was relatively much lower (0.59%). Similarly, moderate malnutrition among the 6–23 months aged children was almost 22%, significantly higher compared to 24–59 months aged children, which was only 4.8%. The cumulative malnutrition rate among the 6–23 months aged children was 25.8% and among the children between 24 and 59 months of age was 5.4%. However, the overall malnutrition among all screened 6–59 months aged children (4502) was 15.2% (Table 3).\n\n\nDiscussion\n\nThe severe malnutrition rates reported in this study are relatively lower compared to figures reported by National Family Health Survey-3 (NFHS 3) (6.8%), which was carried out across the country among the same age group of children. However, the under nutrition rates reported in this study is still high which may have significant negative impact on health, education and productivity of the children. Persistent undernutrition is a major obstacle to human development and economic growth in India, especially among the rural poor and vulnerable areas, where the prevalence of malnutrition is the highest9. Illiteracy, poor health seeking behaviour, unavailability of health care services and poor infrastructure might be other contributing factors of malnutrition among these tribal populations.\n\nThe advantage of using the MUAC measurement compared to other nutritional indices is that it is simple to use and it is good to identify the high risk children who need urgent treatment, facilitating the better coverage at the screening and/or diagnostic stage, which is a key component of program success11. The revision of the MUAC cut off by WHO to identify severe malnutrition is useful in early diagnosis in less severe state of malnutrition whereby it reduces the duration of treatment in therapeutic feeding centres6.\n\nThe government of India is implementing various nutritional interventions including ICDS (Integrated Child Development Services) to address the malnutrition problem among children9. The ICDS program is a well designed and well placed program to address the child undernutrition in the country. However there was more emphasis on coverage rather than on the quality of the program which resulted in limited impact in addressing the malnutrition problem9. Hence, it is necessary that the current ICDS program focuses on improving the quality of tools used to fight the persistent malnutrition among the under-five years old children.\n\nFaulty feeding practices negatively affect the children’s nutritional status, and the current nutrition programs have been unable to make much progress in dealing with these serious issues11. We believe that public health interventions for severe malnutrition must simultaneously focus on preventive and promotive aspects, and therapeutic interventions in the community. There is a paucity of local evidence especially in tribal areas which lack clarity about the possible therapeutic protocols to implement community-based management of severe malnutrition. Evidence from other countries may not be relevant to a very diverse and vast country like India. Research organizations and funding agencies need to prioritize the research further and build a valid evidence base to implement community based malnutrition programs.\n\n\nData availability\n\nMSF obtained data pertaining only to orange and red colour measurements, as the purpose of MUAC screening at mobile clinics was to detect only those children who were malnourished enough to be included in ATFP [Ambulatory Therapeutic Feeding Programme]. For children to qualify for this programme their MUAC measurements should be <118 mm. Hence, only orange and red color measurements data were collected. MSF did not record green and yellow colour measurements for the above mentioned reason.\n\n\nEthical considerations\n\nData were obtained from MSF mobile clinic databases and as a retrospective study, ethical clearance was not necessary. We thank MSF for providing such data.", "appendix": "Author contributions\n\n\n\nSBK and IQ conceived and designed the study. MQ, IQ, SA, SBK analysed the data. SBK and IQ interpreted the data. SBK and MQ drafted the article. All authors revised the article and gave the final approval for publication.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nde Onis M, Blössner M: The World Health Organization Global Database on Child Growth and Malnutrition: methodology and applications. Int J Epidemiol. 2003; 32(4): 518–526. PubMed Abstract \| Publisher Full Text\n\nUNICEF. Picture in India-Nutrition. The children. 2014. Accessed 07/10/2014, 2014. Reference Source\n\nWHO. WHO Expert Committee on Physical Status: The Use and Interpretation of Anthropometry. Technical Report Series No. 854. Switzerland: World Health Organization (WHO). 1995. Reference Source\n\nMei Z, Grummer-Strawn L, de Onis M, et al.: The development of a MUAC-for-height reference, including a comparison to other nutritional status screening indicators. Bull World Health Organ. 1997; 75(4): 333–41. PubMed Abstract \| Free Full Text\n\nFernández MÁ, Delchevalerie P, Van Herp M: Accuracy of MUAC in the detection of severe wasting with the new WHO growth standards. Pediatrics. 2010; 126(1): e195–e201. PubMed Abstract \| Publisher Full Text\n\nWHO/UNICEF. WHO Child Growth Standards and the Identification of Severe Acute Malnutrition in Infants and Children: A Joint Statement by the World Health Organization and the United Nations Children’s Fund. Geneva: World health organization (WHO). 2009. 9241598166. Reference Source\n\nMyatt M, Duffield A: Weight-for-height and MUAC for estimating the prevalence of acute undernutrition? Report for IASC, UNICEF. 2007: 104–110. Reference Source\n\nMSFH. Activity Report 2012–13. New Delhi: Medecins Sans Frontiers Holland India (MSFH India). 2013.\n\nGragnolati M, Shekar M, Das Gupta M, et al.: India’s undernourished children: a call for reform and action. 2005. Reference Source\n\nBriend A: Use of MUAC for severe acute malnutrition. Paper presented at: CMAM forum. 2012. Reference Source\n\nArnold F, Parasuraman S, Arokiasamy P, et al.: Nutrition in India. National Family Health Survey (NFHS-3) India 2005–06. 2009. Reference Source\n\nGoossens S, Bekele Y, Yun O, et al.: Mid-upper arm circumference based nutrition programming: evidence for a new approach in regions with high burden of acute malnutrition. PloS One. 2012; 7(11): e49320. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "6499", "date": "28 Oct 2014", "name": "Samiran Bisai", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe present research work is very important in an Indian context, highlighting the major public health issue where large numbers of malnourished children reside here and the majority of the malnourished children are found among socially and economically underprivileged communities. Tribal population in India is considered as socio-economically underprivileged. However, I have a few minor suggestions as under:The present study is clinic based data collected from two states. Re-analysed data separately for each state. There is large interstate variation of malnutrition and culture as well as food variation. Therefore individual tribal specific data is more important. If available include morbidity history in relation to malnutrition. There are a large number of studies highlighting the application of MUAC as screening tool of undernutrition. More importantly it is a low cost technology as compared to measurement of height and weight. It is to be highlighted in the present manuscript. Compare the prevalence with other studies conducted among tribal population in India.", "responses": [] }, { "id": "6654", "date": "06 Nov 2014", "name": "Kaushik Bose", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe present research work is very useful in highlighting the major public health problem of undernutrition among tribal children of India.Modification required:Re-analyse and compare the prevalence data separately for each state, since there may exist inter-state variation.", "responses": [] }, { "id": "28113", "date": "07 Dec 2017", "name": "James A Berkley", "expertise": [ "Reviewer Expertise Malnutrition", "infectious diseases", "global health" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an important topic and data collected in field operations can be potentially informative to practitioners in India and elsewhere. The main results are given as the prevalence of moderate and severe malnutrition determined by MUAC at 7 mobile clinics in an area affected by chronic insurgency. The paper would be considerably strengthened by a more detailed description of research question being asked, the population and how they were sampled. As currently written, no data on treatment and its outcomes are given. Without these, it will be difficult to draw policy-relevant conclusions.\n\nIn the Introduction, it would be helpful to clarify some of the terminology for readers:\nIn ‘However a higher cut off point of 115 mm was recommended later by WHO as it allows to identify a more accurate number of infants and children with severe acute malnutrition…’, please say what is meant by ‘accurate’. It is known that MUAC and WHZ identify different sets of children with variable overlap. Neither are a true gold standard, but my understanding is the 110cm was a closer match to the previous NCHS WHZ reference, and 115mm is a closer match to the 2006 WHO reference.\n\nSimilarly, in ‘here is large body of evidence strongly suggesting that MUAC is a better indicator of acute malnutrition…’, please give in more detail what is meant by ‘better’. The strongest means of validation is association of anthropometry with subsequent mortality, where MUAC generally performs better than WHZ. Other considerations in community or emergency settings are: reliability of measurement in the field (a few publications have compared MUAC and WHZ); cost, robustness and portability.\n\nBesides WHO, please state what the Indian national malnutrition guidelines recommend.\n\nPlease finish the introduction by stating the aims of the study.\n\nIn the Materials and Methods, more details of the study population and population they were drawn from are needed:\nPlease state the planned study design, or be clear if it was an analysis of operational data (without a prior specific sample size).\n\nPlease state who were the population targeted by MUAC screening. Was it only amongst self-presenters because of a suspected nutrition or health problem, or attending for vaccination? Why did children attend without health problems? Was community sensitisation done (thus influencing the population to attend, and not representing the usual clinic population)? If not door-to-door, what were the potential biases? Or was it designed to be representative of the community? If so, what fraction of the population were measured?\n\nPlease give some more information about the individual sites, including location (a map would be helpful) and factors relating to food security etc. It would also be helpful to have a couple of sentences explaining the Naxalite problem for unfamiliar readers.\n\nPlease briefly describe training and verification for staff undertaking MUAC.\n\nIn ‘…fibre optic color-coded measuring tapes divided into 2 mm additions…’, should ‘additions’ be ‘divisions’?\n\nPlease mention if the fibre optic tapes have been validated against conventional tapes.\n\nIn Results:\nPlease begin the results with the numbers screened, rather than putting this in the Methods section. then give the median (IQR) for age for orange and red groups, and % female/male.\n\nWere there any missing data or implausible values?\n\nPlease provide a histogram of age rather than table 3 since 6-23m is a very large age band, age 6 month olds are quite different to 23 month olds.\n\nIn place of table 3, please give the breakdown of red and orange results by gender, season and by clinic site. Did prevalence vary by site or season? A figure may be useful to show seasonal variation.\n\nAre there any data on outcomes of treatment? This would make the manuscript much stronger.\n\nIn the Discussion:\nIt is not possible to make comparisons with the NFH without the study design having aimed to collect community rather than clinic-based populations. This needs to be clarified above. If the population is representative of clinic attendees, then this comparison should be deleted.\n\nFor the sentence ‘evidence from other countries may not be relevant to a very diverse and vast country like India.’, please be much more specific. Do the authors mean anthropometry (posing a challenge to the WHO 2006 findings that advantaged children grow the same in different settings); or in needs and responses to treatment? If the latter, then please provide some discussion and references on trials and CMAM programmes, e.g. http://gh.bmj.com/content/1/4/e000144, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4381773/ and http://www.ennonline.net/fex/52/acutemalnutritionindiaschildren to start with.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/3-250
https://f1000research.com/articles/3-249/v1	23 Oct 14	{ "type": "Software Tool Article", "title": "Viewing multiple sequence alignments with the JavaScript Sequence Alignment Viewer (JSAV)", "authors": [ "Andrew C. R. Martin" ], "abstract": "The JavaScript Sequence Alignment Viewer (JSAV) is designed as a simple-to-use JavaScript component for displaying sequence alignments on web pages. The display of sequences is highly configurable with options to allow alternative coloring schemes, sorting of sequences and ’dotifying’ repeated amino acids. An option is also available to submit selected sequences to another web site, or to other JavaScript code. JSAV is implemented purely in JavaScript making use of the JQuery and JQuery-UI libraries. It does not use any HTML5-specific options to help with browser compatibility. The code is documented using JSDOC and is available from http://www.bioinf.org.uk/software/jsav/.", "keywords": [ "JavaScript", "sequence alignment", "protein" ], "content": "Introduction\n\nViewing multiple sequence alignments (MSAs) is a fundamental requirement in the analysis of protein sequences, allowing us to visualize conservation across protein families as well as unusual features of particular sequences. As a result, there are a plethora of tools for viewing MSAs. These range from tools which provide attractive printed outputs, through standalone graphical tools — either operating-system dependent, or independent — to web-based viewers.\n\nTwo of the earliest tools were HOMED1 and MALIGNED2 written for VAX/VMS workstations. Neither seems to be actively maintained or easily available any more. Other early viewers include GeneDoc3, BioEdit, Seaview4 and DCSE5 which is part of the RnaViz package for visualizing RNA secondary structures6, but which can be used for protein sequence alignments. A problem in writing graphical software is the operating-system dependency of many graphics libraries. CINEMA7 was probably the first sequence alignment viewer and editor implemented in Java, a platform independent programming language allowing graphical user interfaces (GUIs) to run on any operating system. It has now been rewritten in C++ and is part of UTOPIA8. Other software includes MPSA9, ANTHEPROT10 and ClustalX11, a GUI for the ClustalW multiple sequence alignment program, providing an integrated environment for aligning sequences and analyzing results. Clustal Omega is the most recent version, but at the time of writing only has a command line interface — a beta version of a GUI is due to be released soon.\n\nMore recent developments include the Protein Family Alignment Annotation Tool (PFAAT)12 designed specifically for family analysis and incorporating residue annotation tools as well as integration with Jmol for protein structure display. Like early versions of CINEMA, PFAAT is implemented in Java for operating system independence. CLC Viewer is a recent free package written in Java which contains a number of integrated tools and acts as a core product for adding other features through a commercial version. A more complete list of MSA viewers is available on the web at http://en.wikipedia.org/wiki/List_of_alignment_visualization_software.\n\nProbably the most popular of the available tools is Jalview13 which is available in two versions: a standalone Java application which provides many tools and facilities, and as a ’light’ version (JalviewLight) — a Java applet that can be embedded in a web page. The latter responds to the need for web site developers to be able to embed MSA visualization.\n\nHowever, in recent years there has been a gradual move away from using Java applets in web development. Java creates an additional layer of software (including additional memory consumption) and modern browsers enforce much more caution in running Java applets to avoid security threats. This can result in user irritation with having to accept various pop-up warnings and/or configure security settings, often having to repeat this process when there is a software update. It is not uncommon for Java applets simply to fail to run, perhaps because users do not understand what settings need to be changed. New HTML features such as the HTML5 Canvas, and powerful JavaScript libraries such as Bootstrap, JQuery and JQuery-UI that provide an easier syntax for accessing elements of a web page together with new widgets such as sliders and drag-and-drop support, have overtaken Java as the method of choice for creating interactive web sites with complex requirements. Such features are used widely by popular web sites such as Google Mail, Google Docs, Twitter and Facebook. Illustrating this trend, the Jmol structure viewer has recently been reimplemented in JavaScript as JSmol (http://sourceforge.net/projects/jsmol/). Consequently, over the last couple of years, a small number of JavaScript-based sequence and alignment viewers have started to be developed. These include MODalign14, Alignment-Annotator15, SnipViz16, and Sequence17, a component of the BioJS library18. In addition, there is an intention to port JalviewLight to JavaScript. The available programs are briefly reviewed:\n\nMODalign is part of the MODexplorer package19, a web site for protein modeling, but does not appear to be available as a download for use in other web sites.\n\nAlignment-Editor is part of a more complex system, STRAP. It uses a Java server-side interpreter, Alignment-to-HTML20, which parses the STRAP scripting language and creates an alignment in a form that can be rendered in Web browsers. The server-side element allows tasks such as sequence retrieval, computation of alignments and communication with BioDAS-servers. The rendering system includes a selection of coloring schemes, highlighting of conserved and variable positions in the alignment, reordering and deletion of sequence by drag-and-drop, and residue annotation as well as links to 3D visualization and sequence groups. It exploits JavaScript and HTML5 using the HTML5 canvas to draw helices and other visual elements. However the JavaScript visualizer does not appear to be available by itself. The description of Alignment-Editor15 suggests that alignments should be prepared using the full Java system and the final alignment can then be downloaded as HTML files in a ZIP archive. The software is licensed under the GPL and available from the authors on request, or a desktop version of STRAP can be downloaded or run using Java WebStart. While in principle possible, no simple documentation is provided to enable the client-side JavaScript/HTML5 viewer to be used without the server side software.\n\nSnipViz is a compact and lightweight component designed for display of multiple versions of gene and protein sequences — i.e. essentially identical sequences with mutations. It provides a very simple clean display focused around both DNA and protein sequences allowing very long sequences through a scrolling mechanism which also shows a small box on a representation of the complete sequence to show the relative position within the complete alignment. It also allows display of phylogenetic trees stored in Newick format. Note that SnipViz should not be confused with SNPViz21.\n\nSequence is a BioJS component for visualizing sequences rather than alignments. It only provides very simple views with no choices of coloring schemes, although it does provide very flexible highlighting of regions within a sequence.\n\nJSAV (JavaScript Sequence Alignment Viewer) is a novel JavaScript component that adds to this list. The primary motivation for implementing a new tool was for development of our abYsis antibody database (http://www.abysis.org/), where we required a simple JavaScript component that would enable us to display a set of aligned sequences, sort and select sequences in that alignment for further analysis, and highlight regions of the alignment corresponding to the CDR loop regions of antibodies. Consequently the requirements were as follows: (i) a very simple-to-use lightweight component that can easily be dropped into a web site; (ii) provision of flexible coloring schemes and ’dotifying’ alignments (replacing repeated residues with dots); (iii) the ability to sort sequences — based both on complete sequences or regions of sequences (such as a CDR loop or framework region of an antibody); (iii) the ability to remove sequences from the alignment; (iv) the ability to highlight regions in the alignment and to display a consensus sequence; (v) the ability to export a selected set of sequences in FASTA format; (vi) the ability to submit a selected set of sequences to another web site or to client-side JavaScript code for further processing. Table 1 lists the availability of these and other features in different tools.\n\nNote that the MODAlign web site was unavailable at the time of writing so capabilities have been judged purely on what is published in the paper. † Not available for use in other web sites. ‡ Should be possible, but not designed to be used in this way. * Submission to Modeller only. § Automatically highlights mutated residues. ¶ Highlights columns of residues conserved at a specified level rather than displaying a consensus.\n\n\nSoftware tool\n\nJSAV allows the end-user to modify the display in a number of ways. The web site provider has control over which of these is available to the end user. First, the sequences can be sorted — the code selects the most representative sequence, displaying that at the top of the alignment followed by the most similar sequence and so on. By default, sorting is performed across the whole sequence, but a two-handled slider allows the range of positions on which the sort is based to be modified. Different coloring schemes are available duplicating those provided in Jalview. The alignment can also be ’dotified’, replacing residues repeated between sequences with dots in order to emphasize amino acid differences. Coloring of dotified residues can also be switched off or on. Sequences can be selected and deleted from the alignment; a consensus sequence can be displayed at the bottom of the alignment and updates automatically when sequences are deleted. The complete set of sequences, or a selected subset, can be submitted to another web site, or passed to another JavaScript function for integration with other tools. Tooltips are provided for each option and all options are documented in detail on the web site.\n\nJSAV is implemented purely in JavaScript. Code is managed using GitHub (http://www.github.com) and documented using JSDOC (http://usejsdoc.org). JSAV employs the JQuery library to ease access to elements of the HTML that it generates and uses JQuery-UI to implement a two-value slider that is used to specify a range of positions in the alignment. As input, the code requires an array of JavaScript objects which contain two elements: a unique identifier for a sequence and the sequence itself — all sequences must be pre-aligned. Secondly a set of options can be provided. Options fall into two classes: those that control the (initial) display and those that control facilities available to the end user of a web site to modify the view of the MSA. Options that control the display include: (i) ranges of alignment column positions to be highlighted; (ii) the color scheme to be used; (iii) whether the sequence should be dotified and whether repeated residues should be colored; (iv) whether a consensus sequence should be displayed; (v) whether a FASTA export button should be available and the label for that button; (vi) the URL and label for a button to allow selected sequences to be submitted to another web site; (vii) a JavaScript function name and label for a button to allow selected sequences to be processed by code written by the web site developer; (viii) whether plain tool tips should be used rather than those provided by JQuery — more attractive tooltips available with the ’tooltipster’ package are also supported. Options that control how the end-user can manipulate the display include: (i) whether the alignment should be sortable and, if so, the width and height of the slider used to select a region for sorting; (ii) whether selection check-boxes should be displayed next to each sequence; (iii) whether sequences can be deleted from the alignment; (iv) whether the user should be able to toggle the dotifying of the alignment; (v) whether the user should be able to toggle not coloring dotified residues; (vi) whether a pull-down should be displayed to select color schemes.\n\nThe sequence alignment is rendered as a table and all display of colors and layout is achieved through Cascading Style Sheets (CSS). Consequently, a web-site developer can easily add a new color scheme by modifying the CSS file and setting an option to specify available color scheme names. The number and size of sequences in the MSA is limited only by the memory available to the web browser. A brief extract of sample code is shown in Figure 1 with the results shown in Figure 2.\n\n\n\nJSAV deliberately avoids making use of HTML5 to maximize browser compatibility. It is known to work with modern browsers including Firefox V32.0, Chrome V37, Konqueror V4.13.3, Safari V5.1.7 and Explorer V10.0 on Linux, Mac and Windows platforms and is known to work on versions of Firefox as old as V9.0.1. JSAV has been developed and tested using JQuery V1.10.2 and JQuery-UI V1.10.4, but it only uses the JQuery HTML element selection mechanism and tool tips and the two-handled slider component from JQuery-UI and consequently would be expected to work with much earlier versions.\n\n\nConclusions\n\nViewing multiple sequence alignments is a fundamental requirement of protein sequence analysis. With that large amounts of Bioinformatics work being performed over the web, there is a clear need to be able to embed MSA viewers within web pages. The recent move away from Java in favor of JavaScript has driven a need for MSA viewing tools written in JavaScript. While four other tools have been made available, two do not appear to be available as simple components that can be used by a web developer to provide sequence alignments (MODalign and Alignment-Editor). Of the other two, SnipViz has only very limited facilities and is designed for viewing SNPs in alignments of very similar sequences while Sequence is only designed for displaying single sequences and not MSAs.\n\nConsequently JSAV fills this gap, providing a very simple-to-use component that can just be passed an array or pre-aligned sequences, but which also has the flexibility to allow manipulation of the way in which the MSA is displayed. JSAV provides a number of features that appear to be absent from any of the other tools including dotifying alignments, automatic sorting of sequences (including limiting the sort to a region within the MSA), and submission of selected sequences to other web sites or to other JavaScript code.\n\nFuture directions are likely to include modifying the JSAV component to become part of BioJS18 and linking JSAV with JSMol for structure visualization.\n\n\nSoftware availability\n\nThe software may be downloaded from http://www.bioinf.org.uk/software/jsav/ where demonstrations, including the ability to upload your own MSA, are available together with full documentation implemented with JSDOC.\n\nhttp://www.github.com/AndrewCRMartin/JSAV\n\nhttp://www.dx.doi.org/10.5281/zenodo.1198022\n\nGNU GPL License. Commercial licences available on request.", "appendix": "Competing interests\n\n\n\nJSAV was developed under a grant for commercialization of abYsis, a web-based antibody database and analysis platform (http://www.abysis.org/). JSAV is used as part of abYsis in which University College London and the author have a financial interest.\n\n\nGrant information\n\nDevelopment of this software was funded as part of a BBSRC Follow-On grant (BB/K015443/1).\n\n\nAcknowledgements\n\nACRM thanks the UCL Research Software Development team (in particular, Jens Nielsen) for their contributions to JSAV.\n\n\nReferences\n\nStockwell PA, Petersen GB: HOMED: a homologous sequence editor. Comput Appl Biosci. 1987; 3(1): 37–43. PubMed Abstract \| Publisher Full Text\n\nClark SP: MALIGNED: a multiple sequence alignment editor. Comput Appl Biosci. 1992; 8(6): 535–538. PubMed Abstract \| Publisher Full Text\n\nNicholas KB, Nicholas HB Jr: GeneDoc: Analysis and visualization of genetic variation. EMBNEW NEWS. 1997; 4: 14. Reference Source\n\nGaltier N, Gouy M, Gautier C: SEAVIEW and PHYLO_WIN: two graphic tools for sequence alignment and molecular phylogeny. Comput Appl Biosci. 1996; 12(6): 543–548. PubMed Abstract \| Publisher Full Text\n\nDe Rijk P, De Wachter R: DCSE, an interactive tool for sequence alignment and secondary structure research. Comput Appl Biosci. 1993; 9(6): 735–740. PubMed Abstract \| Publisher Full Text\n\nDe Rijk P, Wuyts J, De Wachter R: RnaViz 2: an improved representation of RNA secondary structure. Bioinformatics. 2003; 19(2): 299–300. PubMed Abstract \| Publisher Full Text\n\nParry-Smith DJ, Payne AW, Michie AD, et al.: CINEMA--a novel Colour INteractive Editor for Multiple Alignments. Gene. 1998; 221(1): GC57–GC63. PubMed Abstract \| Publisher Full Text\n\nPettifer SR, Sinnott JR, Attwood TK: UTOPIA-User-Friendly Tools for Operating Informatics Applications. Comp Funct Genomics. 2004; 5(1): 56–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlanchet C, Combet C, Geourjon C, et al.: MPSA: integrated system for multiple protein sequence analysis with client/server capabilities. Bioinformatics. 2000; 16(3): 286–287. PubMed Abstract \| Publisher Full Text\n\nDeléage G, Combet C, Blanchet C, et al.: ANTHEPROT: an integrated protein sequence analysis software with client/server capabilities. Comput Biol Med. 2001; 31(4): 259–267. PubMed Abstract \| Publisher Full Text\n\nThompson JD, Gibson TJ, Plewniak F, et al.: The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997; 25(24): 4876–4882. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJohnson JM, Mason K, Moallemi C, et al.: Protein family annotation in a multiple alignment viewer. Bioinformatics. 2003; 19(4): 544–545. PubMed Abstract \| Publisher Full Text\n\nClamp M, Cuff J, Searle SM, et al.: The Jalview Java alignment editor. Bioinformatics. 2004; 20(3): 426–427. PubMed Abstract \| Publisher Full Text\n\nBarbato A, Benkert P, Schwede T, et al.: Improving your target-template alignment with MODalign. Bioinformatics. 2012; 28(7): 1038–1039. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGille C, Fähling M, Weyand B, et al.: Alignment-Annotator web server: rendering and annotating sequence alignments. Nucleic Acids Res. 2014; 42(Web Server issue): W3–W6. PubMed Abstract \| Publisher Full Text\n\nJaschob D, Davis TN, Riffle M: SnipViz: a compact and lightweight web site widget for display and dissemination of multiple versions of gene and protein sequences. BMC Res Notes. 2014; 7: 468. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGomez J, Jimenez R: Sequence, a BioJS component for visualising sequences. F1000Res. 2014; 3: 52. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCorpas M, Jimenez R, Carbon SJ, et al.: BioJS: an open source standard for biological visualisation - its status in 2014. F1000Res. 2014; 3: 55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKosinski J, Barbato A, Tramontano A: MODexplorer: an integrated tool for exploring protein sequence, structure and function relationships. Bioinformatics. 2013; 29(7): 953–954. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGille C, Birgit W, Gille A: Sequence alignment visualization in HTML5 without Java. Bioinformatics. 2014; 30(1): 121–122. PubMed Abstract \| Publisher Full Text\n\nLangewisch T, Zhang H, Vincent R, et al.: Major soybean maturity gene haplotypes revealed by SNPViz analysis of 72 sequenced soybean genomes. PLoS One. 2014; 9(4): e94150. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMartin ACR: F1000Research/JSAV. Zenodo. 2014. Data Source" }	[ { "id": "6487", "date": "23 Oct 2014", "name": "Christoph Gille", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI tried JSAV in a few browsers including Opera, Chrome and Konqueror and in all browsers the alignment was well displayed.The tool is fast even when alignments with 40 sequences and more are submitted.It is a clean, robust and simple solution for displaying alignments in web pages.With JSAV, it is very easy to include rendered alignments in web pages.Innovative is the sequence sorter which allows sorting sequences by local sequence similarity.The row header runs out of the screen if the alignment is scrolled horizontally. Also there is no wrapping of long sequences as in Jalview. This matters only in case of sequences comprising more than about 100 residues.Sequence and residue annotations known from Jalview, Pfaat and CLC seem not to be implemented in JSAV.There are some minor issues concerning the GUI: The leading greater-than signs from the fasta header is written in front of the sequence name. The check-box Do-not-color-dots could be deactivated if the check-box Dotify is not activated. The sequences are selected/deselected for deletion or export using check-boxes. Multiple selection of a range of sequences via Shift-left-click does not work. For alignments comprising only a few sequences, however, this does not matter.The text is well written and the advantage of JSAV over other tools becomes clear: It is a simple Java script library for basic alignment visualization which does not require any other server program.The table 1 compares existing tools and is very informative. Interesting is the last table row telling that SnipViz allows long sequences. This could be discussed more in the text since this exemplifies a general performance problem of JS compared to C++ or Java: I assume that in all cited JS based tools with the exception of SnipViz, document-object-model-elements (DOM elements) are created for all residues. For large alignments, ten thousands of elements need to be created which takes long time and uses up much memory. With Java, however, only the visible part of the alignment needs to be drawn (Cinema, Pfaat, Strap) which is much faster and has a minimal footprint. I assume that SnipViz only renders the visible portion of the alignment? This would explain why the alignment cannot be scrolled softly. This kind of discussion would help the reader to understand that using JS for alignment visualization is still technical challenge and one cannot expect the full functionality of CLC workbench or Jalview in a JavaScript library like JSAV.Alignment-Editor should read \"Alignment-Annotator\". This tool should have a mark in the table rows Phylogenetic-tree and Submission-to-another-site.The authors emphasized the program feature of annotations in various cited tools and could provide a row in the table for this important feature.It would be great if annotations could be implemented in future releases of JSAV.", "responses": [] }, { "id": "6666", "date": "12 Nov 2014", "name": "David Martin", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSoftware technical notes are useful contributions to the literature and the state of the art but difficult to review. One ends up assessing the functionality of the software and how readily it can be used for the purpose intended. Does it afford a useful extension to the state of the art, or can it mislead? Can it be readily implemented in workflows or websites (in this case).In general this is a very welcome contribution. The software is easy to incorporate and provides a useful way to visualise sequence alignments. Having said that, there are minor quibbles, the most serious of which is the display of dotted residues. This is of interest where there are a few specific changes and makes them very easy to spot. However, the implementation in this software appears to be somewhat counter-intuitive. The usual method is for differences to a consensus to be shown, with sequences that are identical represented by the dots. In the implementation in the paper, the dots refer to it being the same as the residue above, where all the meaning is lost if colour is not shown. That is somewhat of a caveat emptor, but care should be taken to ensure that such a representation matches the convention expected by the user.It would be useful also to include some form of export for a visualisation, either through a direct export or through integration with a more powerful package such as Jalview.In summary it is a useful tool for representation of short alignments and as such will no doubt find utility, I can certainly see uses for it.", "responses": [] }, { "id": "6667", "date": "03 Dec 2014", "name": "Robert Paul Davey", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI, Robert Davey, promise:i) to not hide behind a screen of anonymityii) to be open and honest with you (the authors) at all timesiii) to be constructive in my criticismiv) within the rules given to me by the journal, to assist you in every way I ethically can to get your manuscript published, by providing criticism and praise that is valid and relevant---The manuscript describes JSAV, a web-browser based multiple sequence alignment viewer that provides similar but expanded features to previous work in this domain.The tool's API and documentation is relatively clear and simple, and the source code is freely available. It also uses standard libraries such as jQuery to provide a consistent experience across browsers, which is a good sign for compatibility and user experience.The authors have given a coherent summary of previous tools, including quite old and standalone solutions for completeness. The authors even go as far as to note potential confusion in similarly named tools (\"Note that SnipViz should not be confused with SNPViz.\") which is a welcome attention to detail.The compatibility argument is sound, but did the authors attempt to develop an HTML5 version of JSAV? What would be the benefits over pure Javascript? In a similar vein, JSAV seems to be responsive and functional with a relatively small number of sequences, and therefore could well be a very useful component for online MSA browsing, but the authors do not mention cases whereby far more complex MSA processing may hit the limits of what JSAV can provide. Tools such as JalView are able to harness \"heavy lifting\" aspects of the Java language. Where do JSAV's abilities end and a desktop tool's features begin? How might JSAV approach these issues?Specific comments:Some sentences are a little long and unwieldy, and could benefit from some reworking, e.g. \"New HTML features such as the HTML5 Canvas, and powerful JavaScript libraries such as Bootstrap, JQuery and JQuery-UI that provide an easier syntax for accessing elements of a web page together with new widgets such as sliders and drag-and-drop support, have overtaken Java as the method of choice for creating interactive web sites with complex requirements.\" The sentence \"have started to be developed\" is clumsy. Consider \"are being developed\", or \"have been developed\". The tools in question have been released. The list of display options (\"Options that control the display include\") produces an overly long paragraph of text. Consider changing this to an indented numbered/bullet list or a table for ease of consumption. \"Bioinformatics\" mid-sentence should not be capitalised. \"The recent move away from Java in favor of JavaScript\" - this sentence should be qualified with a context. Java is very much still used for web application backends, but as the authors previously state, Javascript has become the most popular language recently for client-side rendering and processing.", "responses": [] } ]	1	https://f1000research.com/articles/3-249
https://f1000research.com/articles/3-182/v1	05 Aug 14	{ "type": "Research Article", "title": "Brain-to-Brain (mind-to-mind) interaction at distance: a confirmatory study", "authors": [ "Patrizio E. Tressoldi", "Luciano Pederzoli", "Marco Bilucaglia", "Patrizio Caini", "Pasquale Fedele", "Alessandro Ferrini", "Simone Melloni", "Diana Richeldi", "Florentina Richeldi", "Agostino Accardo", "Luciano Pederzoli", "Marco Bilucaglia", "Patrizio Caini", "Pasquale Fedele", "Alessandro Ferrini", "Simone Melloni", "Diana Richeldi", "Florentina Richeldi", "Agostino Accardo" ], "abstract": "This study reports the results of a confirmatory experiment testing the hypothesis that it is possible to detect coincidences of a sequence of events (silence-signal) of different length, by analyzing the EEG activity of two human partners spatially separated when one member of the pair receives the stimulation and the second one is connected only mentally with the first.Seven selected participants with a long friendship and a capacity to maintain focused mental concentration, were divided into two groups located in two different laboratories approximately 190 km apart. Each participant acted both as a “stimulated” and as a “mentally connected” member of the pair for a total of twenty sessions overall.The offline analysis of EEG activity using a special classification algorithm based on a support vector machine, detected the coincidences in the sequence of events of the stimulation protocol between the EEG activity of the “stimulated” and the “mentally connected” pairs.Furthermore the correlation of the power spectra of the five EEG frequency bands between each of the twenty pairs of data was analyzed using a bootstrap procedure.The overall percentage of coincidences out of 88 events was 78.4% and the statistically significant average correlations between the EEG alpha and gamma bands among the pairs of participants, which confirmed the results observed in a pilot study, support the hypothesis that it is possible to connect two brains and hence two minds at distance.", "keywords": [ "brain-to-brain interaction", "entanglement", "support vector machines" ], "content": "Introduction\n\nBrain-to-brain interaction (BBI) at distance, that is, outside the range of the five senses, has been demonstrated by Pais-Vieira et al., (2013), by connecting the brains of rats via an internet connection.\n\nA similar effect has been demonstrated with humans in a pilot study by Rao & Stocco, (2013) by sending the EEG activity generated by a subject imagining moving his right hand via the internet to the brain of a distant partner which triggered his motor cortex causing the right hand to press a key.\n\nEven though there is cultural resistance in accepting the possibility of observing similar effects in humans without an internet connection, some evidence of these effects nevertheless exists. A comprehensive search of all studies related to this line of research has revealed at least eighteen studies from 1974 until the present time (see Supplementary Material).\n\nIn all these studies the principal aim was to observe whether the brain activity evoked by a stimulus (e.g. by presenting light flashes or images) in one member of a couple, could also be observed in the brain of the partner. Even if some of these studies, those using functional neuroimaging, can be criticized for potential methodological weaknesses that could account for the reported effects (Acunzo et al., 2013), the questions is still open as to whether or not it is possible to connect two human brains at distance.\n\nThe possibility of connecting the brains of two humans at distance without using any classical means of transmission is theoretically expected if it is assumed that two brains, and consequently two minds, can be entangled in a quantum-like manner. In quantum physics, entanglement is a physical phenomenon that occurs when pairs (or groups) of particles interact in ways such that the measurement (observation) of the quantum state (e.g. spin state) of each member is correlated with the others, irrespective of their distance without apparent classical communication.\n\nAt present, generalizability from physics variables to biological and mental variables can be done only by analogy given the differences in their properties, but some theoretical models are already available. For example in the Generalized Quantum Theory (Filk & Römer, 2011; Von Lucadou & Romer, 2007; Walach & von Stillfried, 2011), “entanglement can be expected to occur if descriptions of the system that pertain to the whole system are complementary to descriptions of parts of the system. In this case the individual elements within the system, that are described by variables complementary to the variable describing the whole system, are non-locally correlated”.\n\nReasoning by analogy, we hypothesized the possibility of entangling two minds, and consequently two brains as complementary parts of a single system and studying their interactions at distance without any classical connections.\n\nIn a pilot study, Tressoldi et al., (2014) tested five couples of participants with a long friendship and a capacity to maintain a focused mental concentration, who were separated by a distance of approximately five meters without any sensorial contact. Three sequences of silence-signal events lasting two and half minutes and one minute, respectively, were delivered to the first member of the pair. The second member of the pair was simply requested to connect mentally with his/her partner. A total of fifteen pairs of data were analyzed. By using a special classification algorithm, these authors observed an overall percentage of correct coincidences of 78%, ranging from 100% for the first two segments silence-signal, to approximately 43% in the last two. The percentages of coincidences in the first five segments of the protocol were above 80%. Furthermore a robust statistically significant correlation was observed in all but beta EEG frequency bands, but was much stronger in the alpha band.\n\nThese preliminary results of the pilot study prompted us to devise this pre-registered replication study.\n\n\nMethods\n\nIn line with the recommendations to distinguish exploratory versus confirmatory experiments (Wagenmakers, 2007; Nosek, 2012), we pre-registered this study in the Open Science Framework site (https://osf.io/u3yce).\n\nSeven healthy adults, five males and two females, were selected for this experiment. Their mean age was 35.5, SD = 8.3. Inclusion criteria were a friendship lasting more than five years and their experience in maintaining a focused mental concentration resulting from their experience (ranging from four to fifteen years) in meditation and other practices to control mental activity, e.g. martial arts practices, yoga, etc.\n\nParticipation inclusion followed the ethical guidelines in accordance with the Helsinki Declaration and the study was approved by the Ethics Committee of Dipartimento di Psicologia Generale, prot.n.63, 2012, the institution of the main author. Before taking part in the experiment, each participant provided written consent after reading a brief description of the experiment.\n\nAd-hoc software written in C++ for Windows 7, designed by one of the co-authors, SM, controlled the delivering of the choice of the protocols of stimulation and the timing of the EEG activity recordings of the two partners. EEG activity was recorded by using two Emotiv® EEG Neuroheadsets connected wirelessly to two personal computers running Windows 7 OS. The Emotiv® EEG Neuroheadset technical characteristics are 14 EEG channels based on the International 10–20 locations (AF3, F7, F3, FC5, T7, P7, O1, O2, P8, T8, FC6, F4, F8, AF4, plus 2 references), one mastoid (M1) sensor acts as a ground reference point to which the voltage of all other sensors is compared. The other mastoid (M2) is a feed-forward reference that reduces external electrical interference. Sampling rate is 128 Hz, bandwidth 0.2–45 Hz with digital notch filters at 50 and 60 Hz. Filtering is made by a build in digital 5th order sinc filter and connectivity is obtained by a proprietary wireless connection at the 2.4 GHz band.\n\nOne auditory clip was delivered binaurally at a high volume (80 dBs) to one of the partners through Parrot ZIK® headphones connected with the PC controlling the stimulus delivery and EEG recordings. This clip, reproducing a baby crying, was selected among the list of the worst sounds (Cox, 2008) in order to enhance the EEG activity of the stimulated person.\n\nIn contrast to the pilot study, the stimulation protocol consisted of three different sequences of 30 seconds of listening to the auditory clip interspersed by silent periods lasting one minute (in the pilot study the durations were twice this length). The three sequences comprised 3, 5 and 7 segments (i.e. silence-signal-silence-signal-silence-signal-silence) and were selected by a random algorithm using the rand function of C++ (in the pilot study only a sequence of 7 segments was used). To prevent any possible prediction of the start of the sequence, the duration of the first silence segment was also randomized from one to three seconds.\n\nWe devised a procedure aimed at recreating a real situation when there is an important event to share, in this case a communication relating to a baby crying. In order to isolate the two partners, four of them were located in a laboratory of the Department of General Psychology of Padova University and the remaining three were placed in the EvanLab a private laboratory located in Florence, approximately 190 km away. A research assistant was present at each location.\n\nThe partner designated as “sender” received the following instructions: “when ready, you must concentrate in silence for one to three minutes to relax and prepare to receive the stimulation to send to your partner. To facilitate your mental connection with him/her, you will see a photo of his/her face via the special glasses (virtual glasses model Kingshop OV2, see Figure S1 in the Supplementary Material). Your only task is to endeavor to send him/her mentally what you will hear, reducing your body and head movements in order to reduce artifacts. You will hear a sequence of a baby crying lasting 30 seconds, separated by one minute intervals. The experiment will last approximately 10 minutes”.\n\nThe instructions to the second partner designated as “receiver” were: “when ready, you must concentrate in silence for one to three minutes to relax and prepare to receive the stimulation sent by your partner. To facilitate your mental connection with him/her, you will see a photo of his/her face via the special glasses. Your task is to connect with him/her mentally attempting to receive the stimulation he/she is hearing, reducing your body and head movements in order to reduce artifacts. The experiment will last approximately 10 minutes”.\n\nAfter both partners gave their approval to begin the experiment, the main research assistant located in the EvanLab, started the experiment by informing the second research assistant connected via the internet to trigger the software controlling the experiment. At the end of the experiment, both partners were informed that it was over. After a break, the partners reversed their roles if available.\n\nPairing each participant located in one laboratory with each participant located in the second laboratory, a total of 22 pairs of data were collected, because two participants contributed to only three sessions. Two pairs of data were eliminated due to a faulty recording of the EEG activity.\n\n\nData analysis\n\nThe BrainScanner™ classification software was originally developed and is available from one of the co-authors P.F. (Pasquale Fedele p.fedele@liquidweb.it). The analysis was carried out offline taking the two files of each pair of participant obtained by the Emotiv® EEG Neuroheadset as the input. The first analysis was a classical principal component analysis (PCA) to reduce the data obtained by the fourteen channels to their latent variables. Fifty percent of these data, randomly sampled together with their corresponding labels related to signal and silence were used for the training of the C-supported vector classification (C-SVC) machine (Steinwart & Christmann, 2008; Chang & Lin, 2011).\n\nSupported vector machines (SVMs) are an example of generalized linear classifiers also defined as maximum margin classifiers because they minimize the empirical error of classification maximizing the margins of separation of the categories. SVMs can be considered as alternative techniques for the learning of polynomial classifiers very different to the classical techniques of neural networks training.\n\nNeural networks with a single layer have an efficient learning algorithm, but they are useful only in the case of linearly separable data. Conversely, multilayer neural networks can represent non-linear functions, but they are difficult to train because of the number of dimensions of the space of weights, and because the most common techniques, such as back-propagation, allow to obtain the network weights by solving an optimization problem not convex and not bound, consequently it presents an indeterminate number of local minima (Basheer & Hajmeer, 2000). The SVM training technique solves both problems: it is an efficient algorithm and is able to represent complex non-linear functions. The characteristic parameters of the network are obtained by solving a convex quadratic programming problem with equality constraints or box type (in which the value of the parameter must be maintained within a range), which provides a single global minimum. Regarding the kernel choice, the one that gave the best performance during the pilot tests was the RBF (radial basis function). After the training phase, the algorithm was ready to generalize the obtained classification model to all the data matching the sequence of events of the stimulation protocol with the EEG activity. The result was a contingency table (see examples in Supplementary Table S1) matching the events (silence-signal) with the events detected in the EEG activity of the person mentally connected.\n\nThe EEG activity of each pair was analyzed off-line using the BrainScanner™ classification algorithm, detecting the number of coincidences and the number of errors and missing classifications. Given our interest in detecting the sequence of events (silence-signal) and not their absolute overlap, a signal detected in the EEG activity of the receiver was considered as a coincidence if at least one of its boundaries (initial or final) overlapped with that of the sender (see examples in Figure 1).\n\nThe first row of each example shows the timing and the sequence of periods of silence and stimulation as delivered to the “sender” brain. The second row shows the timing and the sequence of the periods of silence and stimulation identified by the BrainScanner™ classifier in the “receiver” brain. Red color = silence; Black color = signal. Using the criteria to consider a coincidence a segment of the protocol with at least one timing boundary (initial or final) overlapped between the two rows, 6 coincidences can be counted in the first example, 5 in the second and 7 in the third one.\n\nTo check the reliability of the scoring system, the data were analyzed independently by two co-authors, PE and SM. Their overall agreement was 89.3%; discrepancies were solved re-checking the original data. All the individual raw data and results are available for independent analyses at http://figshare.com/articles/BBI_Confirmatory/1030617.\n\nTo have convergent evidence of the relationship between the EEG activity of the two partners, we correlated their EEG activity related to the signal and silence periods recorded in the fourteen channels, with respect to the five frequency bands, delta, theta, alpha, beta and gamma normalized with respect to the total power. Each period of silence and stimulation was divided into tracts of 4 seconds and the Power Spectral Density (PSD) was computed by the periodogram method. The five spectral bands were distinguished as follows: delta (0.5–4 Hz), theta (4–8 Hz), alpha (8–15 Hz), beta (15–30 Hz) and gamma (30–60 Hz). The PSD of the different bands was then expressed in normalized units by dividing the power in each band by the sum of the powers in all the bands.\n\nTo test the significance of the correlation coefficient we adopted a distribution-free approach, the bivariate non-parametric bootstrap (Bishara & Hittner, 2012) with 5000 iterations. From the sampling distribution, we computed the 95% confidence interval following the percentile method. The bivariate test rejects the null hypothesis if r = 0 is not included within the confidence interval. The results are reported in Supplementary Table S1. The raw data and the software source code in MatLab “Accardo_Confirmatory_rev.m” are available at http://figshare.com/articles/BBI_Confirmatory/1030617.\n\n\nResults\n\nThe numbers of coincidences in the EEG activity in the pairs of participants detected by the BrainScanner™ classifier, related to the three different stimulation protocols in the twenty sessions are reported in Table 1a, Table 1b and Table 1c.\n\nThe expected number of coincidences of the signal events was zero, whereas the expected number of coincidences of silence events was 100%. A percentage of coincidences of the signal events well above what would be expected by chance (i.e. 50%), can be a demonstration of a brain (mind) connections between the pairs of participants unless statistical or procedural artifacts can explain them.\n\nThe overall percentages of coincidences and their precision were estimated with the corresponding confidence intervals. Furthermore the Bayes Factor comparing the hypothesis that the percentage of coincidences will outperform the percentage of errors and missing data with the hypothesis of null difference between these two percentages, was calculated with the online applet available at http://pcl.missouri.edu/bf-binomial, using a uniform prior probability distribution based on a beta distribution.\n\nThe classification algorithm correctly detected 69/88 (78.4%; 95% CI: 68.7–85.7) events, 26/34 (76.4%; 95% CI: 58.4–87.5) related to the signals and 43/54 (79.6%; 95% CI: 67.1–88.2) related to the silence events.\n\nThe corresponding Bayes Factors comparing the H1 (above chance detection) vs H0 (chance detection) hypothesis, for the overall and the signal coincidences are 390625 and 27.1 respectively.\n\nIt is interesting to observe that for all three stimulation protocols, the percentages of coincidences of the first three events (silence-signal-silence) was 98.3%, dropping to 40.9% for the next two events (signal-silence) and to 16.6% for the last two events (signal-silence). This drop was also observed in the pilot study, even if it was less dramatic: 83.3% and 43.3%, respectively. However it is important to recall that in the pilot study, the duration of the signals and the silence periods were 60 seconds and 180 seconds, respectively. A plausible explanation of this difference can be the limitation of the present version of our classifier to extract sufficient information to differentiate the two classes of events from the EEG activity, postulating that the signal/silence ratio of EEG activity reduced after a sequence of three events.\n\nThe Pearson’s r correlation values with corresponding 95% CIs between the silence and signal events of each of the twenty pairs of participants separately for the five frequency bands, are reported in the Table S1 (see Supplementary Material). The corresponding graphs are available at http://figshare.com/articles/BBI_Confirmatory/1030617.\n\nIn Figure 2, we report the alpha band normalized power spectrum values recorded in the fourteen channels of the EEG activity of pair 15 as an example of strong correlation.\n\nLegend: T = Transmitter; R = Receiver.\n\nThe average correlations among the twenty pairs estimated with 5000 bootstrap resamplings with the corresponding confidence intervals for each EEG frequency band, separately for the silence and signal events, are reported in Table 2.\n\nStatistically significant correlations are colored in bold.\n\nAs observed in the pilot study, we found reliable correlations in the alpha band for both silence and signal events and in the gamma band only for the silence events. In the pilot study we also observed the strongest correlation in the alpha band.\n\nFourteen out of the twenty pairs of participants showed statistically significant correlations in at least one of these two frequency bands.\n\nCompared with the pilot study of Tressoldi et al., (2014), in the present study the pairs of participants were approximately 190 km away each other, the length of the sequence of events was randomized and the durations of the silence and signal periods were reduced. However, the percentage of the overall correct sequences of events and the correlation between the EEG frequency bands of the pairs of participants observed in this confirmatory study were almost identical with those observed in the pilot study. In the pilot study, the overall percentage of correct identification of the events was 78%; 95% CI=72–87 with respect to the 78.4%; 95% CI=68.7–85.7, observed in the present study.\n\nFurthermore the average correlation estimated with 5000 bootstrap resamplings among all pairs of data was 0.58; 95% CI=0.46–0.69 and 0.55; 95% CI=0.43–0.65 for the alpha band respectively for the silence and the signal periods in the pilot study and 0.32; 95% CI=0.18–0.44, for silence and 0.27; 95% CI=0.13–0.40, for signal events in this confirmatory study. For the gamma band, the correlation values were 0.36; 95% CI=0.24–0.49 and 0.32; 95% CI=0.19–0.46 for the silence and signal, respectively, in the pilot study and 0.23; 95% CI=0.10–0.37 and 0.12; 95% CI=-0.009–0.26 in the present study.\n\nThe differences in the strength of correlations between the pilot and the present study may well be explained by the reduction of fifty percent in the duration of the silence and signal events with a consequent increment of the signal/noise ratio.\n\nThe alpha band is a marker of attention (Klimesch et al., 1998; Klimesh, 2012), whereas the gamma band is a marker of mental control as typically observed during meditation (Lutz et al., 2004; Cahn et al., 2010) and in this case the correlations we have observed could represent an EEG correlate of the synchronized attention between the pairs of participants.\n\nWe think that these results are mainly due to the innovative classification algorithm devised for this line of investigation and the enrolment of participants selected for their long friendship and experience in maintaining a mental concentration on the task. The drop of coincidences after three segments, corresponding to approximately five minutes, could be a limit of our classification algorithm to detect the differences between silence and signal, because of an increase of exogenous and endogenous EEG noise correlated to fatigue and loss of concentration (mental connection) between the two partners.\n\nAre these results sufficient to support the hypothesis that human minds and their brains, can be connected at distance? Only multiple independent replications can support this hypothesis both using our data and data obtained using different participants.\n\nWhich artifacts could explain our results? The large distance between the pair of participants excludes any sensorial connections between them. The only possibilities of artificial connections between the EEG activity of the pairs of participants could be caused by sensorial triggers sent to the participant with the role of “receiver” by the computer recording his/her EEG activity. This possibility was excluded because the randomization, both of the length of the first silence period and of the length of sequences of events, was controlled only by the computer connected with the EEG activity of the participant with the role of “transmitter” and no acoustic or visual events were associated with these computations. Another possible source of artifacts could derive from the research assistants managing the computers connected with the EEG activity of the two participants. In this case the only possibility of synchronizing the EEG of the two participants could be obtained if the research assistant who randomized the type of the sequence of events sent this information to the research assistant of the “receiver” who sent auditory signals to influence the EEG activity of the “receiver”. All our research assistants were part of the research team and this possibility can be excluded with certainty.\n\nCould our results be simply artifacts obtained by the software we used to analyze the data? This is an open question that could be solved using different classifiers and by analyzing the software we used for the correlations.\n\nWhile awaiting new and independent controls and replications of our findings, we are planning to improve the current stimulation protocol to support a simple mental telecommunication code at distance. For example, it is sufficient to associate any small sequence of events with a message, i.e. silence-signal = “CALL ME”; silence-signal-silence = “DANGER”, etc.\n\nThe next steps of this line of research are an optimization of the classification algorithm to detect longer sequences of events and the analysis of data online.\n\n\nData availability\n\nfigshare: BBI_Confirmatory, doi: http://dx.doi.org/10.6084/m9.figshare.1030617 (Tressoldi, 2014a).\n\nThe BrainScannerTM classification software used in this study is available on request from Pasquale Fedele, email: p.fedele@liquidweb.it.\n\nThe ad-hoc software written in C++ for Windows 7 used to control the delivery of the choice of protocols and the timing of the EEG activity recordings is available under a CCBY license from figshare: Mind Sync Data Acquisition Software, doi: http://dx.doi.org/10.6084/m9.figshare.1108110 (Tressoldi, 2014b).", "appendix": "Author contributions\n\n\n\nPE, LP, AF, PC, SM devised the experiment; MB, PF and AA contributed to the software development; PE, LP, AF, PC, SM, DR and FR contributed to the data collection, PE and LP wrote the paper.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was funded by Bial Foundation contract 121/12.\n\n\nAcknowledgements\n\nThe English was revised by the Proof Reading Service.\n\n\nSupplementary material\n\nThe results of a comprehensive search of all studies related to this line of research revealed at least eighteen studies from 1974 until the present time. These references are presented in a Word document as part of the Supplementary Material.\n\nValues in bold are statistically significant (when the confidence intervals do not include the zero).\n\n\nReferences\n\nAcunzo DJ, Evrard R, Rabeyron T: Anomalous experiences, psi and functional neuroimaging. Front Hum Neurosc. 2013; 7: 893. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBasheer IA, Hajmeer M: Artificial neural networks: fundamentals, computing, design, and application. J Microbiol Methods. 2000; 43(1): 3–31. PubMed Abstract \| Publisher Full Text\n\nBishara AJ, Hittner JB: Testing the significance of a correlation with nonnormal data: comparison of Pearson, Spearman, transformation, and resampling approaches. Psychol Methods. 2012; 17(3): 399–417. PubMed Abstract \| Publisher Full Text\n\nCahn BR, Delorme A, Polich J: Occipital gamma activation during Vipassana meditation. Cogn Process. 2010; 11(1): 39–56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChang CC, Lin CJ: LIBSVM: a library for support vector machines. ACM TIST. 2011; 2(3): 27. Publisher Full Text\n\nCox TJ: Scraping sounds and disgusting noises. Appl Acoust. 2008; 69(12), 1195–1204. Publisher Full Text\n\nFilk T, Römer H: Generalized quantum theory: Overview and latest developments. Axiomathes. 2011; 21(2): 211–220. Publisher Full Text\n\nKlimesch W: α-band oscillations, attention, and controlled access to stored information. Trends Cogn Sci. 2012; 16(12): 606–617. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKlimesch W, Doppelmayr M, Russegger H, et al.: Induced alpha band power changes in the human EEG and attention. Neurosci Lett. 1998; 244(2): 73–76. PubMed Abstract \| Publisher Full Text\n\nLutz A, Greischar LL, Rawlings NB, et al.: Long-term meditators self-induce high-amplitude gamma synchrony during mental practice. Proc Natl Acad Sci U S A. 2004; 101(46): 16369–16373. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNosek BA: Open Science Collaboration. An open, large-scale, collaborative effort to estimate the reproducibility of psychological science. Perspect Psychol Sci. 2012; 7(6): 657–660. Publisher Full Text\n\nPais-Vieira M, Lebedev M, Kunicki C, et al.: A brain-to-brain interface for real-time sharing of sensorimotor information. Sci Rep. 2013; 3: 1319, 1–. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRao R, Stocco A: Direct Brain-to-Brain Communication in Humans: A Pilot Study. 2013. Reference Source\n\nSteinwart I, Christmann A: Support vector machines. Springer, 2008. Reference Source\n\nTressoldi P: BBI_Confirmatory. figshare. 2014a. Data Source\n\nTressoldi P: Mind Sync Data Acquisition Software. figshare. 2014b. Data Source\n\nTressoldi PE, Pederzoli L, Caini P, et al.: Brain-to-brain (mind-to-mind) interaction at distance: a pilot study. 2014. Publisher Full Text\n\nVon Lucadou W, Romer H: Synchronistic phenomena as entanglement correlations in Generalized Quantum Theory. J Consc Stud. 2007; 14(4): 50–74. Reference Source\n\nWagenmakers EJ, Wetzels R, Borsboom D, et al.: An agenda for purely confirmatory research. Perspect Psychol Sci. 2012; 7(6): 632–638. Publisher Full Text\n\nWalach H, von Stillfried N: Generalised quantum theory—basic idea and general intuition: a background story and overview. Axiomathes. 2011; 21(2), 185–209. Publisher Full Text" }	[ { "id": "5767", "date": "12 Aug 2014", "name": "James Lake", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review and comment on this important paper. This unique pilot study provides a strong beginning case for direct brain-to-brain communication. Further research along these lines incorporating advanced EEG analysis and brain-computer interface (BCI) technologies will lead to important insights about mechanisms underlying innate human Psi abilities while also developing practical approaches that will enhance the human capacity to both ‘detect’ and ‘send’ discrete units of ‘information’ at a distance.The protocol used to create a maximally ‘arousing’ stimulus in the brain of the stimulated person is a novel approach to Psi research based on the (reasonable) assumption that the sound of a crying baby is very distressing to most humans and thus may represent a ‘maximally arousing’ stimulus. In a future study it would be interesting to measure EEG activity in a matched control group to determine whether or in what ways background EEG activity may change when the control group is exposed to the recorded sound of a baby crying. I believe it would add rigor to your methodology to develop a ‘normative’ data set of EEG responses to the stimulus (ie, through recordings from a representative cross-section of different ages and genders, etc) and to compare this ‘normative’ response to findings from the pilot study and future research findings.I am interested to know how you decided on the stimulus duration and interval between stimuli. I am also interested to know how you determined the total duration (15 minutes). Were you concerned with subject fatigue? Did you arrive at these parameters from insights gained during the training phase? I am curious as to whether you might have obtained more robust correspondences using either a longer stimulus duration or a longer experiment duration. Your comments on this would be very useful. In terms of the algorithm of checking EEG data between ‘sender’ and ‘recipient’ for apparent above-chance correspondences, did you examine data during simultaneous recording epochs only, and did you consider examining the data for possible correspondences off-set in time? There appears to be an implicit assumption in your methodology that any significant correspondences between 2 (or more brains) will take place simultaneously or in the same general time ‘period.’ Along these lines it would be interesting to design statistical methods that could test for a time 'displacement' effect that may take place in your model of direct brain-to-brain communication.I did not completely understand the goals of the ‘training phase’ or how this is done. It would be helpful to have a clearer and more detailed description of this part of the study. My understanding is that subjects are initially ‘trained’ to achieve robust responses to the ‘baby cry’ stimulus after which they enter into the experiment so that it will then be more likely that they will experience EEG responses of the type your algorithm is designed to detect. By extension, if the EEG response following stimulation by the ‘baby cry’ sound is robust and consistent across subjects, is your assumption that this stimulus would more likely ‘evoke’ a distant ‘signal’ in the receiver? In this case is ‘training’ really about trying to optimize/maximize signal to noise ratio in the ‘distant signal’ that may entangle two (or more) brains following the baby cry stimulus?If I understand correctly, the classification algorithm built into the “BrainScanner” software comprises the critical aspect of your study in that the strength of your findings rests on the assumption that the correspondences in EEG activity obtained using the BrainScanner software and the Emotiv Neuroheadset are equivalent to real-time changes in EEG activity between two or more subjects monitored in parallel. Along these lines it would be helpful to clarify why the algorithm built into the “BrainScanner” software is the most appropriate algorithm for the study goals you have in mind. A more complete explanation of this point would also help me better understand your arguments for validating the significance of findings of apparent correspondences between the selected stimulus (ie, sound of baby crying) and general or specific changes in EEG activity in the ‘receiver.’Regarding the kind of information extracted from raw data, does the algorithm compare measures of discrete simple parameters like measures of amplitude, frequency and wavelength or more complex dynamic measures, for example cordance? Along these lines, if cordance is taken into account, does the algorithm analyze cordance with respect to mean differences between two or more brain regions? Does the algorithm average over different frequency or time domains, if so what are the parameters and threshold values and how are these derived and defended in developing your methods? Along these lines, it would be helpful in the discussion section to comment on the observation of ‘apparent correspondences’ between EEG traces in two or more humans because of common EEG dynamics, and in to include remarks that artifacts and shared EEG dynamics have been ruled out providing a compelling argument that findings cannot be ‘explained away’ by these phenomenon.One potential concern along these lines is that the built-in software algorithm may be removing or simplifying particular EEG features prior to analysis which may in turn result in the appearance of greater uniformity and therefore the appearance of above-chance correspondences between two or more EEG tracings. In future studies it will be important to employ EEG apparatus and filtering software that directly address the possibility of apparent correspondences as an artifact of automated EEG filtering prior to analysis. The question concerns whether, in the absence of the software algorithm imposing ‘structure’ on more primitive signals, there would still be a statistically significant correspondence between two (or more) simultaneous recordings from two or more brains. Another phenomenon that that may give the appearance of significant real-time correspondences where none are present, and that should be directly addressed in future studies, has to do with the finding that a significant component of average EEG activity reflects widely shared EEG dynamics among the majority of humans in other words EEG activity between two or more humans probably varies in non-random ways. Such patterned non-random EEG activity may result in the appearance of correspondences when two or more brains are monitored. This issue has been raised in previous critiques of EEG (and fMRI) Psi studies. In order to build a compelling case that your findings do not incorporate bias and to verify a Psi effect you may first need to provide strong evidence ruling out artifacts or apparent correspondences between unique EEG traces due to widely shared EEG dynamics, as both may lead to the appearance of above-chance correspondences. This will require careful analysis of the recording method used, assumptions built into the software algorithm, and statistical methods used to test for significance.", "responses": [ { "c_id": "939", "date": "12 Aug 2014", "name": "Patrizio Tressoldi", "role": "Author Response", "response": "Thank you for the accurate review and all comments to improve the present version. We will address all of them in the new version after we will receive the responses of the other referees.For the moment we will reply to only some of the comments.\"...how you decided on the stimulus duration and interval between stimuli.\"Before defining the present stimulation protocol, we ran multiple pilot trials. The goal was to identify a protocol simple to implement, short in duration (to avoid boredom and fatigue to the participant) and with sufficient signal to noise gain to be detected by our equipment. In the first formal pilot experiment, cited in the paper, we tested officially a first version of the protocol and the positive results prompted us to improve that protocol. On pag. 7 in the General Discussion we discuss these differences. \"I did not completely understand the goals of the ‘training phase’ or how this is done\"The training phase is not for the participants but only for the BrainScanner classifier. As for most classifiers, it is necessary to train the software to identify the best algorithm (both linear and nonlinear) to discriminate the two (in our case) class of EEG activity (silence and signal). After this phase, the software apply this algorithm to all data. We will clarify better this point in the next version. \"In order to build a compelling case that your findings do not incorporate bias and to verify a Psi effect you may first need to provide strong evidence ruling out artifacts or apparent correspondences between unique EEG\"In the new version we will expand this point. For the moment it is important to consider that the converging evidence supporting a real B-to-B interaction at distance is based on a replication of the results obtained in the pilot study even if using a shorter protocol of stimulation and the correlations in the alpha e gamma bands. If one looks at the graph of each single pair (available here), there are striking similarities in almost all pairs." }, { "c_id": "995", "date": "18 Sep 2014", "name": "Patrizio Tressoldi", "role": "Author Response", "response": "Did you examine data during simultaneous recording epochs only, and did you consider examining the data for possible correspondences off-set in time?Reply: At present we do not have any plausible hypothesis about the possibility of a time-displacement effect, hence we are focusing our efforts to demonstrate a simultaneous correlation....it would be helpful to clarify why the algorithm built into the “BrainScanner” software is the most appropriate algorithm for the study goals you have in mind...does the algorithm compare measures of discrete simple parameters like measures of amplitude, frequency and wavelength or more complex dynamic measures, for example cordance?Reply: BrainScanner is one of the different classifiers available (see Amancio et al., 2014) . These classifiers are one of the best options to classify signals in two or more categories. In our case for the input we used the results of the principal component analysis of the raw data, but it is possible to use other EEG characteristics as the input..... it would be helpful in the discussion section to comment on the observation of ‘apparent correspondences’ between EEG traces in two or more humans because of common EEG dynamics, and in to include remarks that artifacts and shared EEG dynamics have been ruled out providing a compelling argument that findings cannot be ‘explained away’ by these phenomenon........ The question concerns whether, in the absence of the software algorithm imposing ‘structure’ on more primitive signals, there would still be a statistically significant correspondence between two (or more) simultaneous recordings from two or more brains........you may first need to provide strong evidence ruling out artifacts or apparent correspondences between unique EEG traces due to widely shared EEG dynamics, as both may lead to the appearance of above-chance correspondencesReply: Some of these concerns were also shared by Dr Schwarzkopf. Our use of the raw data without any preprocessing or filtering and the fact that correspondences were observed only using those precise parameters (50% of random data from all silence and signal segments for training the classifier), and after five replications, offer a cautionary optimism about the reliability of our results. Moreover, in the Discussion we expanded the section related to possible artifacts. However if all agree that more experiments by us and independent authors are necessary both to confirm our findings and exclude alternative explanations of a nonlocal interaction." } ] }, { "id": "6065", "date": "09 Sep 2014", "name": "Sam Schwarzkopf", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study aims to test the rather unusual hypothesis that the brains of two individuals separated geographically by almost 200 km can form a telepathic link that is measurable with EEG. While this is arguably an implausible hypothesis, it is certainly testable. Unfortunately, I believe this is not an adequate experiment to test such a hypothesis. There are major problems both with the experimental design and the statistical analysis. While the authors may be able to address some of my comments with additional analyses and control experiments, the sheer number of issues demands a complete overhaul of the entire study. I broke up my concerns into three sections: 1. Non-naive participants and predictability of the protocol: There were only 7 participants in this experiment. This means that in order to collect the 20 data sets (ignoring 2 excluded ones) every person participated in multiple recording sessions, both as “sender” and as \"receiver\" (except for one person (subject A) who acted only once as sender but thrice as receiver). Therefore even if participants were naive about the experimental protocol before their first recording session, in subsequent sessions they would be very familiar with both the arousing auditory stimulus (sound of a crying baby) and with the sequence of events (30s stimulus/signal periods interspersed by 60s periods of silence). In fact, the “senders” were even explicitly told about this sequence at the start of each session. Moreover, typically the roles of “sender” and “receiver” were reversed for a pair of participants right after their first recording. Thus the knowledge of the design would have been fresh in the second “receiver’s” mind. On this note, based on the subject initials in the data spreadsheets I wonder if the first author participated in this experiment? This is not explicitly stated so perhaps it is simply a coincidence that the initials match. All of the other subject initials also match those of other authors, however, so at the very least it should be acknowledged who (if any) among the participants were authors. Of course, it can be entirely acceptable to take part in your own study but this should always be reported in the methods and it very much depends on the experimental design. Certainly, for a study of this kind, where the predictability of experimental events is critical, I would be very concerned how an in-depth knowledge of the experimental protocol affects the results. It would certainly make the claim somewhat questionable that participants could not have known the randomisation of the protocols. Perhaps the biggest improvement over the pilot experiment, in which the sequence and duration of the stimulus protocol was always fixed and thus completely predictable, is the fact that the overall duration of the protocol was randomised (from three options, i.e. protocols with 1, 2, or 3 stimulus periods) and that the duration of the initial silence period was apparently randomised between 1-3 minutes (but see point 1.4). Thus the ‘receiver’ should have been less able to predict the exact onset of the first stimulation period. However, after this initial onset the protocol was always fixed (i.e. 30s stimulus periods separated by 60s silence), so provided they could make a reasonable guess about the onset of the first stimulus period, the rest of the session would still have been rather predictable. Inspection of the traces of the stimulus protocols suggests that for the three different protocols the sequence of stimulation events was always perfectly aligned with the onsets of several other protocols. This is because the randomization of the initial silence period was not somewhere between 1-3 minutes as implied in the methods (actually this states “seconds”, but the corresponding author already acknowledged that this is a typo). Instead, as far as I can tell the initial silence period appears to have been either 1 min or 2 min. This of course means that the onset of the first stimulus period was either been at 1 min (12/20 sessions) or 2 min (8/20 sessions). As discussed in point 1.3, after this onset the sequence would have been fairly predictable to the participant. The duration of the initial silence was therefore the most unpredictable part of the experiment for the “receiver” – provided they had no other cues (see point 1.5) or had prior knowledge of the randomisation (see points 1.2 and 1.6). Is it conceivable that there were any cues for the “receiver” whether this session had 1 min or 2min of initial silence? It is unclear from the description of the methods whether the picture of the “sender” appeared in the goggles from the beginning of the recording session, including the initial silence period (but I assume this was the case). What could the participants hear and feel from the experimental room, noises made by the attending research assistants, etc? Assuming that the timestamps on the spreadsheets indicate the timing of recording, it can be seen that for two thirds of the pairs, the duration of the initial silence period in the second recording was the opposite of that in the first recording. Since these pairs were just reversing the role of “sender” and “receiver”, the “receiver” could then be predisposed to expect a shorter/longer initial silence period in the second recording compared to the first. So even if “receivers” always assumed that the onset of the stimulus was the opposite of the first session they would have been correct more than half of the time (see also my discussion of incorrect statistical assumptions in point 3). Moreover, blocks of sessions typically had one participant in common (sessions 1-4 subject F, sessions 5-10 subject D, sessions 11-13 subject A, 14-30 subject PT). It thus seems likely that “receivers” were implicitly aware of the randomisation of the onsets. Regarding the predictability of the sequence, is it possible that there were any time cues helping the “receiver” keep the timing and thus predict the sequence of stimulus events? While the participants were wearing headphones and goggles, could they have heard the ticking of a clock or a dripping tap or other regular noises (perhaps from the experimental equipment)? Could there have been signals in other sensory modalities (floor vibrations, air flow in the room)? Such cues need not even be external for the participants could have kept time, e.g. by using their respiration. In particular considering that all participants had experience with meditation, yoga, or similar practices this does not seem unrealistic. As discussed, the only aspect of the experiment that was comparably unpredictable (except for the potential caveats discussed in the previous points) was the onset of the initial silence period. Subsequently, the sequencing of stimulus and silence was fixed and it was actually fairly unimportant whether the overall protocol duration was short (1 stimulus), medium (2 stimuli), or long (3 stimuli) because participants would only have to maintain the fixed rhythm of 30s stimulus followed by 60s silence. The fact that decoding becomes progressively worse for segments later in the protocol (as shown by Tables 1a-c) may thus be a result of the “receiver’s” inability to maintain the rhythm as the session progressed. This is in part supported by some of the traces in which the classifier detected stimulus periods that considerably exceeded 30s in the latter half of the session (in particular, session “tLrPT”). The deterioration of decoding accuracy could also be due to the uncertainty as to whether there would be more stimulus periods or not because the participant could not be sure that they were in a short, medium, or long session. In summary, there were multiple problems with familiarity of participants with the experimental paradigm and the predictability with the rhythm of stimulus and silence periods. To address this, the experiment should have been made much more unpredictable with properly randomized onsets and jittered durations for all the silence events.\n\n2. The nature of the decoded signal: The participants’ familiarity with the crying baby stimulus also raises further questions. First, it somehow undermines the whole idea of transmitting information between two brains. Except for the first session all participants already knew that the stimulus was a crying baby. This makes transmitting that information redundant. More importantly, it also means that participants could have been imagining (or at least thinking about) the crying baby sound at regular intervals prescribed by the experiment. In that case, the classifier algorithm would have simply decoded the thoughts/imagery or the mental effort of the \"receiver\" to receive the crying baby sound. Combined with the issues with predictability of the sequence discussed in point 1, this would make the results hardly surprising. One way to address this would be to have two very distinct signal events and training the classifier to distinguish those in addition to the silence period (e.g. a crying baby vs a calming surf). I can however understand that the authors focused only on binary events (stimulus or silence) but in that case at the very least they would have to address the concerns with the predictability of the stimulus sequence discussed in point 1. A related problem with the decoding analysis is that there is no way of knowing whether the decoded signal has anything to do with the “sender’s” experience of a crying baby. Was there any debriefing of participants? Did any of the “receivers” hear a crying baby during the recording session? Or perhaps that is expecting too much. Did they at the very least report the feeling of receiving any information from the sender? One way to control for this would have been to have sessions both with and without a “sender” (obviously randomised so that the “receiver” could not know) and to see if the classifier still identifies stimulus and silence periods at these regular intervals. The previous suggestion would also help to address another concern about the nature of the decoded signal. As the authors themselves (briefly) acknowledge in the discussion, the alpha and gamma frequency bands are markers of attentional engagement, arousal, or mind wandering. Thus the decoding might instead simply exploit the temporal evolution of the EEG signal over the course of the session related to these factors. While this does not entirely explain why the decoding is so high for the first stimulus period (but see discussion of this problem in point 1), it certainly would be an alternative explanation for why decoding becomes progressively worse over the course of the session (see also point 3.5). 3. Incorrect statistical assumptions and questions about analysis: The statistical analysis used for testing whether decoding performance was above chance levels is incorrect, because the authors did not take into account that this is an unbalanced design. Therefore, contrary to the authors’ description in the methods the expected chance level is not 50%. Because stimulus periods made up less of the overall duration than silence periods, even if the classifier consistently (and incorrectly) assigned the silence label decoding accuracy would be greater than 70% (i.e. the proportion of silence within a session). The propensity of the classifier to choose one over another class label is also not necessarily 50%, especially in unbalanced designs. The use of a standard binomial test against 50% chance performance is therefore not correct. Instead the authors should have used a permutation test that estimates the true chance performance under these conditions. The “coincidences” measure used by the authors is also questionable. It is not immediately clear whether the definition of overlapping segments could have inflated the decoding results somehow. It seems odd that this measure was used at all considering that it should be straightforward to compare the traces directly. It also seems strange that there was a subjective disagreement between the two raters – the definition of coincidences sounds pretty simple. The methods do not provide nearly sufficient detail to understand how the decoding analysis was performed. The authors state that they used PCA to reduce the dimensionality of the EEG channels but they do not state what data were actually used for classification. Was it the band-pass filtered EEG signal trace within short time windows? Was it the frequency-power spectrum within each time window? How long were the time windows? Or was a sliding time window used? In this context it is also quite odd that the classifier performs so consistently. It certainly seems very odd that the classifier would hardly ever misclassify the initial silence period or that there are never any gaps within stimulus periods. Physiological data are typically very noisy. It is surprising to see such reliable classification even for the “sender”, let alone the “receiver”. Again, this is difficult to understand without a clear idea of how exactly the classification was performed. It is also unclear what the classifier was trained on. The authors state that a randomly selected “fifty percent of these data” were used for training. Did the authors use 50% of the data for training separately for each participant in the pair and then test the classifier on the remaining 50%? This would be incorrect because there are likely to be temporal correlations between adjacent data points (again, this would be a lot clearer if we knew what data were actually used). Or were these 50% from the “sender” and then used to classifier 100% of data from the “receiver”? It seems more defensible to assume that the “sender” and “receiver” are statistically independent (unless of course the hypothesis of a telepathic link is true). However, the temporal proximity of data points might still be a concern even in this case (see also points 2.3). Especially considering the fact that the authors used one of the most powerful linear SVM kernels (radial basis function) for classification, it is very unclear what attributes in the data the classifier exploited. One of the main problems with such multivariate decoding analyses is in fact that it is entirely opportunistic – the algorithm will find the most diagnostic information about the class labels in the data to produce the an accurate classification without any regard to whether this diagnostic information is actually meaningful to the hypothesis. So without any better understanding of what was done, it is quite plausible that the classification simply decoded how much time had passed since the start of the experiment. One way to reveal this would be to rerun the classification with different class labels that are orthogonal to the stimulation sequence. If the classifier exploited some attribute about the temporal evolution of the signal it should still perform well under those circumstances. The lack of methodological detail also makes it impossible to understand the description of the correlation analyses. It is stated that recordings were broken up into 4s time bins. How does this relate to the correlations that were calculated? In Figure 2 alpha power in different channels are plotted. This does however not indicate which of the periods (i.e. first, second or third stimulus, or average across them?) these power values came from (at least according to my count pair 15 should have had three stimulus periods). It also does not explain whether this is just the data from one of the 4s bins or an entire segment or if it was averaged across all segments? How were the correlations listed in Table 2 averaged across pairs? Was it taken into account that the same participants contributed to several of these correlations? Moreover, was any correction for multiple comparisons applied to the number of frequency bands? Several of the decoding traces for the “receiver” contain zeros. Two examples are in fact shown in Figure 1 in the top and middle rows. What does that mean? Was the recording simply stopped at that point? If so, why? The stimulus protocol with the sender was still running at that time? The authors state that the recording for the “receiver” was triggered manually by the research assistant after receiving the signal via the internet from the lab with the “sender”. Would this not introduce an uncontrolled lag in the recordings? Surely it should be technically feasible to automate this and trigger the recording simultaneously (or at least with a fixed lag due to the internet transmission)?", "responses": [ { "c_id": "973", "date": "11 Sep 2014", "name": "Patrizio Tressoldi", "role": "Author Response", "response": "Many thanks indeed for your accurate review and the time spent on our paper. The reply to all your and the second reviewer comments and a new version of the paper wil be posted ASAP." }, { "c_id": "974", "date": "11 Sep 2014", "name": "D. Sam Schwarzkopf", "role": "Reviewer Response", "response": "After writing this review a number of additional issues have come to my attention. For the review I had merely inspected the decoding traces included in the spreadsheets in the Coincidences folder. However, inspection of the actual raw data files reveals the following problems: Decoding arbitrary stimulus labels1. We loaded a few of the raw data and applied decoding analysis using a non-linear SVM classifier with RBF kernel. As described in my review, the methods provide insufficient detail to understand what was done. Nevertheless, it is easy to replicate almost perfect decoding (98-99% correct) of the stimulus label using what seems to be the procedure described in the manuscript. Critically, however, this works for arbitrarily chosen stimulus labels that are completely independent from the actual stimulation sequence (this is the control analysis I suggested to the authors in point 3.5 of my review). Examples of such decoding analysis can be seen at: http://dx.doi.org/10.6084/m9.figshare.1167456This demonstrates that the decoding has nothing to do with the stimulation sequence or with any purported connection between the brains of Sender and Receiver – rather the classifier exploits temporal correlations in the recorded signal because training and test data are not truly independent. Therefore even if the authors controlled for all of the other concerns I raised in the review, the general analysis approach is simply incorrect for addressing this question. Irregularities in the raw data files2. The time stamps for Sender and Receiver do not match for Pair 3 (4 hours time difference) and Pair 17 (70 minutes time difference). 3. In fact, for Pair 3 the file for the Receiver (subject PT) and the file for the Sender in Pair 16 are exactly identical (except for the stimulus condition labels in column 3). We can speculate that this is due to a copying error and that the Receiver in Pair 3 should have been subject F. 4. For Pair 12 the Sender is listed as subject P but it presumably should have been subject PT - according to the Coincidences files subject P was never paired as Sender with subject A as Receiver. Non-naive participants 5. The subject names in the raw data files essentially confirm that all participants in this experiment were also authors on this manuscript (see point 1.2 in my review). Again, it can be acceptable to include some authors in studies and in some situations it may even be defensible if all participants are authors, especially if the design is truly unpredictable and the experiment studies a very basic process. However, this should be acknowledged explicitly in the methods, and it seems difficult to justify this in an experiment such as this where the participants should be naive with regard to the experimental protocol." }, { "c_id": "980", "date": "12 Sep 2014", "name": "Patrizio Tressoldi", "role": "Author Response", "response": "May you clarify which events you classified with your non-linear SVM classifier? Segments corresponding to the stimulation protocols (i.e. silence 60 sec; signal 30 sec.) or different ones?" }, { "c_id": "984", "date": "15 Sep 2014", "name": "D. Sam Schwarzkopf", "role": "Reviewer Response", "response": "Thank you for your question. I concede that the decoding analysis I performed is very rudimentary. I merely took the raw data observations, that is, columns 4-17 (counting from left) in the raw data spreadsheets are the input data. I understand these are the raw signals from the 14 channels. As labels I either used the stimulus labels in the third column or arbitrarily assigned labels that are uncorrelated with the true stimulus. Following the description in your methods section I then randomly sampled 50% of the observations (rows) in the data and the labels to be used as training set and the other 50% as testing set.This approach works well for decoding the presence or absence of the \"stimulus\" regardless of whether you train and test on the same data set (that is, sender or receiver) or whether using 50% of the sender as training set and the other 50% of the receiver as testing set (the latter shows modestly reduced decoding accuracy). Presumably it should work also when using data from different pairs in particular for those who had identical stimulus protocols. I haven't tried that yet though.It does depend on the length of the \"stimulus\" periods you choose. Using a random stimulus that assigns the stimulus as being on or off for each sample (row) in the data does not work. It does therefore depend on having slow stimulus periods presumably because the classifier exploits slow temporal correlations.The main difference I can see between mine and your analysis (in so far I understood it correctly) is that you first transformed the 14 data columns into eigenvalues using PCA. But the lack of this step cannot explain the artifactual decoding in my reanalysis. PCA merely reduces the dimensionality of the data set by taking into account the covariance between the 14 channels and expressing the data in this lower dimensional space (although you didn't specify how many principal components you used in the decoding). This may in fact explain why the decoding periods are slightly less noisy in your case - although I'm still surprised by that.Or did you use another set of observations, say, short time windows rather than the individual samples as I did here? If so this should be described in more detail as it is far from clear in the present form. Even so, however, any data transformation you may have used would I think preserve the underlying problem: decoding works for arbitrary stimuli. This is why it also isn't surprising that the presumably erroneous data set in Pair 3 can be decoded well even though the wrong subject (sender data from Pair 16) seems to have been used here with the incorrect stimulus labels. The same applies to the pairs which were recorded with large time delays, that is Pairs 17 and 3 (although this time delay may simply be due to the incorrect data file for the receiver).Unlike the first reviewer I think it is entirely premature to speculate about \"brain-to-brain\" links to operate across time - rather I would say that recordings at different times (different days) would serve as excellent control data sets. My prediction, according to what we have seen so far, is that decoding using this method will work very well even across different days and across different subject pairs because it simply relies on the temporal evolution of the slow wave EEG signal under these experimental conditions. To sum up all of my (admittedly very extensive) criticisms, I think the one major overarching problem with this experiment is that it lacks an appropriate control. There is no experimental condition that allows you to pit the telepathy hypothesis against alternative explanations. There simply is no way of knowing whether the decoding has anything to do with the sender, with the task, with the stimulus, etc. Unless I made an enormous oversight, the reanalysis I attempted strongly suggests that decoding has nothing whatsoever to do with the stimulus protocol. Even if decoding works across Sender and Receiver (or even across participants from different pairs) there is no way to rule out that it simply exploits the temporal evolution of the signal, which is presumably similar under these conditions.I know this is harsh, but given these problems I don't see any other way to address these criticisms than to completely redo the experiment using a properly controlled design." }, { "c_id": "986", "date": "15 Sep 2014", "name": "D. Sam Schwarzkopf", "role": "Reviewer Response", "response": "Correction: In my previous comment I said that the decoding works even when training on Sender and testing on the Receiver. This is however not correct. I looked at that analysis briefly but I didn't plot back the decoding. Of course, the classification was >80% correct but that is simply because the classifier in this case incorrectly classifies everything as silence. Chance accuracy in this case is of course exactly at that level. This is in fact the error with incorrectly assuming a chance level of 50% I mentioned in my review and it shows you how easy it is to misinterpret such results!Therefore, the most critical thing you need to clarify is 1) the nature of the data you used to classify, 2) what exactly was chosen as training and test observations, and 3) was this done separately for Sender and Receiver?" }, { "c_id": "994", "date": "18 Sep 2014", "name": "Patrizio Tressoldi", "role": "Author Response", "response": "1. Non-naive participants and predictability of the protocol concerns.Reply: You correctly pointed out the participants were not naïve to the experimental procedure, but this was an intentional selection and in the second version of the paper we disclosed they were all co-authors. The choice of these participants was justified in the Method section. Only future studies will confirm if the same results can be observed including no-selected participants.The matter that they could self-generate a differential EEG activity corresponding to the timing of three protocols of stimulation was your major concern.We think it could be possible if all the following conditions are satisfied:They trained a lot to self-induce a differential EEG modulation activity following the timing of our protocol: one type of EEG activity for 1 min, another type for 30 sec, repeatedly; They were able to synchronize the above EEG activity with the timing of the protocol delivered to the “sender” partner, predicting when it started, after 1,2 or 3 minutes.As to point a) we assure that none of the participants achieved this ability and applied it during the experiment. Furthermore it is the opinion of Dr. Giovanni Mento, an expert in the EEG correlates of time estimation, is that at present there is no evidence a human adult could obtain such ability for time segments above few seconds, in particular because the precision of time estimation worsens with the length of the times to estimate, following the Weber law (e.g. Piras & Coull, 2011). As to point b) the randomization of the stimulation protocol start should predict a correct synchronization for only 33% of the trials.Instead, as far as I can tell the initial silence period appears to have been either 1 min or 2 min. Reply: In the “Stimulation protocol” paragraph, we clarified that the randomization of the onset of the stimulation protocol was among 1,2 or 3 minutes.Is it conceivable that there were any cues for the “receiver” whether this session had 1 min or 2min of initial silence? .......What could the participants hear and feel from the experimental room, noises made by the attending research assistants, etc?Reply: the “receiver” saw the image of the “sender” before the formal start of the stimulation protocol and remained visible until the end of the session. After the PC connected to the EEG was activated, the research assistant remained outside the room where the “receiver” was placed. The sound attenuated lab and the headphones filtered all external noises. No visual or auditory cues were available to be used to predict the sequence of events.Since these pairs were just reversing the role of “sender” and “receiver”, the “receiver” could then be predisposed to expect a shorter/longer initial silence period in the second recording compared to the first.Reply: If participants engaged in an intentional guessing of the exact onset of the stimulation protocol, they could guess correctly 1/3 of the sessions. As clarified in point 1, our selected participants guaranteed the adherence of the instructions.The nature of the decoded signal: .....A related problem with the decoding analysis is that there is no way of knowing whether the decoded signal has anything to do with the “sender’s” experience of a crying baby.Reply: the initial project aimed at comparing implicit EEG activity with explicit reporting by the “receiver”. However we did not find stimulus, both visual and auditory that could be presented for 30 sec without inducing an habituation apart the “baby crying” clip. However we aim at investigating the implicit/explicit reporting differences.Incorrect statistical assumptions and questions about analysis:....the expected chance level is not 50%.Reply: In effect the definition of 50% as the expected chance level is misleading, given that we estimated only the percentage of coincidences overall and for silence and signal segments separately, and computed the Bayes Factor comparing the hypothesis that the percentage of coincidences were above the percentage of errors and missing. The authors state that they used PCA to reduce the dimensionality of the EEG channels but they do not state what data were actually used for classificationReply: in the version 2 we have clarified that we used the raw EEG data of participants with the role of \"receivers\" without any pre-processing or filter.Did the authors use 50% of the data for training separately for each participant in the pair and then test the classifier on the remaining 50%? This would be incorrect because there are likely to be temporal correlations between adjacent data points.......it is quite plausible that the classification simply decoded how much time had passed since the start of the experiment. One way to reveal this would be to rerun the classification with different class labels that are orthogonal to the stimulation sequence. If the classifier exploited some attribute about the temporal evolution of the signal it should still perform well under those circumstances.Reply: In the “Classification algorithm” paragraph we clarified that even if the EEG recording of both the sender and the receiver have been analyzed with the BrainScanner, only the results of the participants in the role of “receiver” were reported. We confirm we used 50% of the data for training the classifier together with the labels defining the silence and signal periods. In the pilot study, we observed that this percentage and the reduction of the signal periods to be analyzed, worsened the classification accuracy. The possibility that with 50% of the data used for the training it may be possible to predict the remaining 50% simply because of a temporal correlation between adjacent points is probabilistically possible if the randomization selected only or the majority of even or odd numbers. Repeating the classification five times we ruled out this possibility.Reversing the classification labels yields the same results, as the task of the classifier is simply to detect differences between the two categories of events if any, but it is “blind” with respect to their meaning or nature...correlation analyses....This does however not indicate which of the periods (i.e. first, second or third stimulus, or average across them?) these power values came fromReply: In the “correlation analysis” paragraph we specified that the normalized Power Spectral Density was calculated by collapsing all silence and signal periods.How were the correlations listed in Table 2 averaged across pairs? ..... Moreover, was any correction for multiple comparisons applied to the number of frequency bands?Reply: The overall correlation results presented in Table 2 are obtained by averaging the correlations presented in Table S1, to take in account individual (pair) differences. For the statistical approach we used, now disclosed in the paragraph “Statistical approach”, familywise error rate controls don't apply.Several of the decoding traces for the “receiver” contain zeros.....What does that mean? Was the recording simply stopped at that point?Reply: In the examples presented in Figure 1 we clarified that these were considered as missing. As explained in the text, these are the outputs of our classifier that used all data of the participants with the role of \"receivers\" recorded applying the three stimulation protocols.Irregularities in the raw data filesReply: Many thanks for the accurate control of our raw data. Now we have uploaded the correct files.I would say that recordings at different times (different days) would serve as excellent control data sets. Reply: we agree that the next step is to devise a control condition where we expect no correlation and coincidences. Apart your suggestion, we could add \"no stimulation\" trials where no visual or auditory information are delivered interspersed with \"stimulation trials\" like those used in the present study. Given we are devising a new pre-registered study, would you agree to have an \"adversial collaboration\" with us?" } ] } ]	1	https://f1000research.com/articles/3-182
https://f1000research.com/articles/3-248/v1	21 Oct 14	{ "type": "Research Article", "title": "Metabolomics in psoriatic disease: pilot study reveals metabolite differences in psoriasis and psoriatic arthritis", "authors": [ "April W. Armstrong", "Julie Wu", "Mary Ann Johnson", "Dmitry Grapov", "Baktazh Azizi", "Jaskaran Dhillon", "Oliver Fiehn", "Julie Wu", "Mary Ann Johnson", "Dmitry Grapov", "Baktazh Azizi", "Jaskaran Dhillon", "Oliver Fiehn" ], "abstract": "Importance: While “omics” studies have advanced our understanding of inflammatory skin diseases, metabolomics is mostly an unexplored field in dermatology.Objective: We sought to elucidate the pathogenesis of psoriatic diseases by determining the differences in metabolomic profiles among psoriasis patients with or without psoriatic arthritis and healthy controls.Design: We employed a global metabolomics approach to compare circulating metabolites from patients with psoriasis, psoriasis and psoriatic arthritis, and healthy controls.Setting: Study participants were recruited from the general community and from the Psoriasis Clinic at the University of California Davis in United States.Participants: We examined metabolomic profiles using blood serum samples from 30 patients age and gender matched into three groups: 10 patients with psoriasis, 10 patients with psoriasis and psoriatic arthritis and 10 control participants.Main outcome(s) and measures(s): Metabolite levels were measured calculating the mean peak intensities from gas chromatography time-of-flight mass spectrometry.Results: Multivariate analyses of metabolomics profiles revealed altered serum metabolites among the study population. Compared to control patients, psoriasis patients had a higher level of alpha ketoglutaric acid (Pso: 288 ± 88; Control: 209 ± 69; p=0.03), a lower level of asparagine (Pso: 5460 ± 980; Control: 7260 ± 2100; p=0.02), and a lower level of glutamine (Pso: 86000 ± 20000; Control: 111000 ± 27000; p=0.02). Compared to control patients, patients with psoriasis and psoriatic arthritis had increased levels of glucuronic acid (Pso + PsA: 638 ± 250; Control: 347 ± 61; p=0.001). Compared to patients with psoriasis alone, patients with both psoriasis and psoriatic arthritis had a decreased level of alpha ketoglutaric acid (Pso + PsA: 186 ± 80; Pso: 288 ± 88; p=0.02) and an increased level of lignoceric acid (Pso + PsA: 442 ± 280; Pso: 214 ± 64; p=0.02).Conclusions and relevance: The metabolite differences help elucidate the pathogenesis of psoriasis and psoriatic arthritis and they may provide insights for therapeutic development.", "keywords": [ "Psoriasis", "metabolomics", "psoriatic arthritis" ], "content": "Introduction\n\nPsoriasis is a chronic, inflammatory skin disease associated with significant morbidity and mortality1. Approximately one-third of psoriasis patients develop psoriatic arthritis2,3. The exact mechanisms underlying psoriasis remain under investigation. A complex interplay between genetic and environmental factors initiates a cascade of events that lead to activation of dendritic cells4. The activated dendritic cells stimulate differentiation and migration of effector T cells (Th1 and Th17) to the skin. The subsequent release of inflammatory cytokines promotes further recruitment of immune cells, stimulates keratinocyte proliferation and sustains a state of chronic inflammation4.\n\nGenomics, proteomics, and transcriptomics research has substantially contributed to the growing body of knowledge concerning psoriasis etiology. For example, genome-wide transcriptional analysis and association studies have identified new susceptibility loci that implicate pathways in psoriasis pathogenesis which integrate epidermal barrier dysfunction with immune dysregulation5–7. A recent proteomic analysis of psoriatic skin tissue identified 36 up-regulated proteins associated with the regulation of cell death, defense response, inflammatory response, and reactive oxygen species8. However, the data beyond genomics and proteomics at the metabolic level can provide new insights into the cellular processes involved in psoriasis.\n\nMetabolomics, the most recent addition to the “omics” fields, is the comprehensive study of all low molecular mass metabolites in the metabolome of an organism under a given set of conditions9. Examining the metabolic byproducts produced at the transcriptional and translational levels will yield clues to the cellular regulatory processes and underlying molecular networks. Because metabolites are the end products and the most downstream representation of cellular processes, the study of an organism’s metabolome is most reflective of any observable phenotype9. Metabolomics analysis will enable researchers to gain valuable information regarding the physiology of a system by measuring the amplified output that results from genetic and environmental perturbations10. To date, metabolomics has not yet been applied to investigate the systemic mechanisms underlying psoriasis.\n\nBecause psoriasis is a multifactorial disease at the genomic, protein, and cellular levels, it is important to examine the end products of cellular processes in psoriasis and psoriatic arthritis. In this novel study, we employed a global metabolomics approach using gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) to compare the serum metabolic profiles of patients with psoriasis, patients with psoriasis and psoriatic arthritis, and healthy controls. We then summarized our results within the context of existing literature to generate hypotheses for future targeted analysis. Integrating genomics, proteomics, transcriptomics and metabolomics data from all levels will add to our understanding of the physiological processes underlying this complex disease and will provide potential insights into its associated comorbidities.\n\n\nMethods\n\nIn this study, we examined the metabolite profiles of psoriasis patients with and without psoriatic arthritis and healthy controls using GC-TOF-MS. Blood serum samples were obtained from 30 patients: 10 patients with psoriasis, 10 patients with both psoriasis and psoriatic arthritis, and 10 healthy control participants. Patient demographics and treatments are summarized in Table 1. Plaque psoriasis is the most common subtype of psoriasis, and the diagnosis made by dermatologists based on characteristic red, scaly patches and plaques on characteristic locations. The Psoriasis Area and Severity Index (PASI) is a validated and widely used outcomes instrument in clinical trials that measures psoriasis severity. Psoriatic arthritis is an inflammatory arthritis that typically affects the peripheral joints but can also have axial manifestations, enthethitis, and dactylitis. In approximately 70–80% of patients with psoriatic arthritis, psoriasis precedes the development of joint symptoms. This study was approved by the Institutional Review Board at University of California, Davis (Protocol 254745-11). The Declaration of Helsinki was followed and the study participants provided their written, informed consent.\n\nThis table describes demographics and clinical information on the patient cohort that contributed their samples to this metabolomics study. Patient demographics indicated for patients with psoriasis (Pso), and patients with psoriasis and psoriatic Arthritis (PsA): age, sex, Body Mass Index (BMI), Psoriasis Area and Severity Index (PASI), duration of condition (in years), and current treatment(s). Demographics of age and sex for control patients are also indicated.\n\nBlood samples were collected from patients using a BD 5 ml double-gel serum separator tube. After 30 minutes of coagulation, the samples were immediately centrifuged at 2000 rpm for 15 minutes to complete blood fractionation11. Thereafter, the sera were transferred to Eppendorf tubes, snap-frozen in liquid nitrogen for 60 s, and stored at -80°C until further processing.\n\nSamples were analyzed using a GC-TOF approach. The study design was entered into the MiniX database12. A Gerstel MPS2 automatic liner exchange system (ALEX) was used to eliminate cross-contamination from sample matrix occurring between sample runs. 0.5 microliter of sample was injected at 50°C (ramped to 250°C) in splitless mode with a 25 sec splitless time. An Agilent 6890 gas chromatograph (Santa Clara, CA) was used with a 30 m long, 0.25 mm i.d. Rtx5Sil-MS column with 0.25 µm 5% diphenyl film; an additional 10 m integrated guard column was used (Restek, Bellefonte PA)13–15. Chromatography was performed at a constant flow of 1 ml/min, ramping the oven temperature from 50°C for to 330°C over 22 min. Mass spectrometry used a Leco Pegasus IV time of flight mass (TOF) spectrometer with 280°C transfer line temperature, electron ionization at −70 V and an ion source temperature of 250°C. Mass spectra were acquired from m/z 85–500 at 17 spectra/sec and 1750 V detector voltage.\n\nResult files were exported to our servers and further processed by our metabolomics BinBase database. All database entries in BinBase were matched against the Fiehn mass spectral library of 1,200 authentic metabolite spectra using retention index and mass spectrum information or the NIST11 commercial library. Identified metabolites were reported if present in at least 50% of the samples per study design group (as defined in the MiniX database); output results were exported to the BinBase database and filtered by multiple parameters to exclude noisy or inconsistent peaks15. Quantification was reported as peak height using the unique ion as default. Missing values were replaced using the raw data netCDF files from the quantification ion traces at the target retention times, subtracting local background noise12. Sample-wise metabolite intensities were normalized by the total signal for all annotated analytes. Daily quality controls and standard plasma obtained from NIST were used to monitor instrument performance over the length of the data acquisition.\n\nStatistical analysis was implemented on log2 transformed metabolite values in R16. The significance levels (i.e. p-values) were adjusted for multiple hypothesis testing according to Benjamini and Hochberg17 at a false discovery rate (FDR) of 5%.\n\nOrthogonal signal correction partial least squares discriminant analysis (O-PLS-DA)18 was carried in out in R16 using the Devium package (https://github.com/dgrapov/devium).\n\nA biochemical and chemical similarity network19 was developed for all measured metabolites with KEGG20 and PubChem CIDs21 identifiers. Enzymatic interactions were determined based on product-precursor relationships defined in the KEGG RPAIR database. Molecules not directly participating in biochemical transformations, but sharing many structural properties, based on PubChem Substructure Fingerprints22, were connected at a threshold of Tanimoto similarity ≥ 0.7.\n\n\nResults\n\nAnalysis of the human sera samples using GC-TOF-MS yielded 144 known metabolic structures and a total of 354 molecular features (Dataset). When comparing the metabolite profiles of psoriasis patients and control patients, the key differences that reached univariate significance were characterized by an increase in alpha ketoglutaric acid (mean peak intensity ± std dev: Pso: 288±88; Control: 209±69; p=0.03), and decreases in asparagine (mean peak intensity ± std dev: Pso: 5460±980; Control: 7260±2100; p=0.02), glutamine (mean peak intensity ± std dev: Pso: 86000±20000; Control: 111000±27000; p=0.02), and beta-sitosterol (mean peak intensity ± std dev: Pso: 217±100; Control: 315±130; p=0.04) in psoriasis patients compared to control patients (Figure 1, Dataset). OPLS-DA showed that all models performed significantly better (Q2=0.42±0.1; RMSEP=0.37±0.1) than what is expected based on random chance (permuted: Q2=-0.51±0.7; RMSEP=0.68±0.1).\n\nBiochemical and chemical similarity networks were used to map statistical and multivariate modeling results within a biological context. The vertices represent metabolites which are connected by edges based on biochemical relationships (yellow, KEGG) and chemical similarities (lavender, structural similarity). Vertex size represents the metabolite's importance and the color represents the direction of statistically significant changes (blue, decrease; red, increase; gray, insignificant p>0.05).\n\nUpon comparing the metabolite profiles of patients with both psoriasis and psoriatic arthritis to patients with skin-limited psoriasis, the top metabolite differences were characterized by a decrease in alpha ketoglutaric acid (mean peak intensity ± std dev: Pso+PsA: 186±80; Pso: 288±88; p=0.02), and increases in arabinose (mean peak intensity ± std dev: Pso+PsA: 624±460; Pso: 363±90; p=0.04), lignoceric acid (mean peak intensity ± std dev: Pso+PsA: 442±280; Pso: 214±64; p=0.02), phosphoric acid (mean peak intensity ± std dev: Pso+PsA: 20600±4300; Pso: 17100±3300; p=0.05), and glycerol-3-galactoside (mean peak intensity ± std dev: Pso: 193±140; Pso+PsA: 367±190; p=0.02) in psoriasis patients with concomitant psoriatic arthritis compared to patients with psoriasis alone (Figure 2, Dataset). The OPLS-DA results suggest that the developed model did not perform significantly better (Q2=0.66±0.2; RMSEP=0.54±0.1) than what is expected based on random chance (permuted: Q2=0.47±0.3; RMSEP=0.55±0.1).\n\nBiochemical and chemical similarity networks were used to map statistical and multivariate modeling results within a biological context. The vertices represent metabolites which are connected by edges based on biochemical relationships (yellow, KEGG) and chemical similarities (lavender, structural similarity). Vertex size represents the metabolite's importance and the color represents the direction of statistically significant changes (blue, decrease; red, increase; gray, insignificant p>0.05)\n\nKey metabolite differences between patients with both psoriasis and psoriatic arthritis and healthy controls are characterized by increases in phosphoric acid (mean peak intensity ± std dev: Pso+PsA: 20600±4300; Control: 16300±4000; p=0.02), glucuronic acid (mean peak intensity ± std dev: Pso+PsA: 638±250; Control: 347±61; p=0.001), arabitol (mean peak intensity ± std dev: Pso+PsA: 238±65; Control: 172±61; p=0.03), and arabinose (mean peak intensity ± std dev: Pso+PsA: 624±460; Control: 322±140; p=0.02) in patients with both psoriasis and psoriatic arthritis compared to healthy individuals (Figure 3, Dataset). OPLS-DA analysis suggested that the developed model performed better (Q2=0.86±0.1; RMSEP=0.50±0.05) than what is expected based on random chance (permuted: Q2=0.48±0.4; RMSEP=0.55±0.1) and displays weak predictive performance.\n\nBiochemical and chemical similarity networks were used to map statistical and multivariate modeling results within a biological context. The vertices represent metabolites which are connected by edges based on biochemical relationships (yellow, KEGG) and chemical similarities (lavender, structural similarity). Vertex size represents the metabolite's importance and the color represents the direction of statistically significant changes (blue, decrease; red, increase; gray, insignificant p>0.05).\n\n\nDiscussion\n\nPsoriasis is widely recognized as a condition that is more than “skin deep”. The infiltration of inflammatory cells in psoriasis dermis and epidermis may be released into systemic circulation, contributing to chronic systemic inflammation23. To our knowledge, this is the first study that employs a metabolomics approach to analyze human serum and examine metabolite changes in circulation beyond the skin. The spectral results indicated differences in low molecular weight compounds that may help distinguish psoriasis patients from healthy controls, psoriasis patients from patients with both psoriasis and psoriatic arthritis, and psoriasis patients with psoriatic arthritis from healthy controls. Although our metabolomics analysis detected a variety of altered metabolite levels, many of which warrant investigation, herein, we discuss the altered levels of the metabolites where the most relevant literature exists. We place our findings within the context of existing literature and explore their relationship to the immune-mediated and inflammatory nature of the disease.\n\nGlutamine. Glutamine, the most abundant free amino acid in the body, is essential for protein synthesis and cellular growth24. In this study, GC-TOF-MS analysis revealed a decrease in glutamine levels in the sera of patients with psoriasis when compared to healthy controls. The alteration in glutamine levels may be due to higher rates of protein synthesis, glutamine consumption by immune cells, and/or differences in transglutaminase levels between patients with and without psoriasis.\n\nPsoriasis is characterized by high rates of cellular proliferation, which increases the cellular demand of amino acids, notably glutamine, to accommodate for the higher rate of protein synthesis. This increased demand for glutamine is analogous to alterations of glutamine levels reported in patients with cancer, a condition also characterized by increased cellular proliferation24,25. In addition, psoriasis is also characterized by a dysregulated immune system whereby the amount and activity of immune cells are enhanced4. The proliferation and functions of immune cells are highly dependent upon glutamine. High rates of glutamine consumption exhibited by lymphocytes, macrophages, and neutrophils have been reported in numerous studies in vitro26,27. Specifically, glutamine consumption enhances the production of many cytokines, and among them, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-1β (IL-1β), and interleukin-6 (IL-6), all of which play an important role in psoriasis innate immunity.\n\nAnother possible explanation for the observed decrease in glutamine levels may be related to the up-regulation and increased expression of keratinocyte tranglutaminase in psoriatic lesions28,29. Keratinocyte transglutaminase is an enzyme critical for cornified envelope formation and participates in epidermal differentiation. Transglutaminase catalyzes the formation of the N-(γ-glutamyl)-lysine isopeptide bond using the amino acids glutamine and lysine. The inhibition of keratinocyte transglutaminase expression by retinoic acid may explain, in part, one of the beneficial effects of retinoid use to treat psoriasis.\n\nAsparagine. Asparagine, a common non-essential amino acid, is the amide of aspartic acid and is easily hydrolyzed during the cell aging process30,31. The spectral results showed a decrease in asparagine levels in the serum of psoriasis patients, which may result from spontaneous asparagine deamidation, a process enhanced by an oxidative microenvironment. Chronic oxidative stress has been reported in patients with psoriasis32. In asparagine deamidation, a spontaneous post-biosynthetic modification, the asparagine residue undergoes nucleophilic attack and forms a succinimide ring intermediate. The succinimidyl residue is an unstable five-membered ring that hydrolyzes to give a mixture of aspartyl and isoaspartyl forms30. The resulting isoaspartate residue (isoAsp) is formed through a beta linkage as opposed to the normal alpha conformation, and is favored 3:1 over the normal conformation at physiological conditions31. The presence of isoAsp residues in proteins has been shown to decrease protein function and can trigger autoimmune responses31. However, a protein repair system that employs the enzyme L-isoAsp-(D-Asp)-O-methyltransferase (PIMT) has recently been identified. PIMT specifically targets the isoAsp residues and forms an unstable methyl ester (L-isoaspartyl methyl ester), which is rapidly converted back to the L-succinimidyl form. The L-succinimidyl residues are then hydrolyzed to the L-aspartyl form30. This repair system prevents the accumulation of damaged proteins.\n\nThe observed decrease in asparagine is consistent with the findings from a study by D’Angelo et al. (2012), who examined the abnormal isoaspartyl residues in the erythrocyte membranes of psoriasis patients33. D’Angelo et al. found that L-isoAsp content was highly increased in the red blood cells (RBC) membrane proteins of psoriatic patients. By measuring methyl esterification through an in situ protein methylation approach, the investigators found that protein methyl esterification was increased by 1.5 fold in psoriatic patients compared to controls. To determine whether these data are due either to an increase in deamidation rate or to an impaired repair system in patients with psoriasis, they measured the intracellular levels of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). D’Angelo et al. found that the transmethylation potential (SAM/SAH) was comparable in patients and controls, indicating a normal repair system in psoriasis patients. Therefore, the increase in methyl esterification is more likely to be attributed to increased protein instability at asparagine sites, which is responsible for asparagine degradation and IsoAsp accumulation in psoriasis patients. Of note, isoAsp residues may confer immunogenicity to previously ignored self-antigens and need to be further explored given the immune-mediated nature of psoriasis31.\n\nAlpha ketoglutaric acid. Alpha ketoglutarate is an intermediate of the citric acid cycle involved in cellular metabolism. The increase in alpha ketoglutaric acid levels detected in serum of psoriasis patients may be due to enhanced alpha ketoglutarate synthesis. Alpha ketogluarate is synthesized from isocitrate through a process mediated by isocitrate dehydrogenase. Enzymatic assays from previous studies have reported significantly increased enzymatic activities of isocitrate dehydrogenase in psoriasis patients34.\n\nThe increase in alpha ketoglutaric acid levels may be a contributing factor to the increase in cellular proliferation associated with psoriasis. Singh et al. (2013) and Vishnoi et al. (2013) found that the in vitro addition of alpha ketoglutarate greatly enhanced cellular proliferation rates, cell survival, and metabolic activity. Alpha ketoglutarate functions as a scavenger for removing toxic metabolites within cells, thereby enhancing cell viability. Additional studies support alpha ketoglutarate involvement in cellular proliferation through regulation of the mTOR-signaling pathway35. mTOR, a protein kinase responsible for cell proliferation, cell survival, and protein synthesis, is phosphorylated by alpha ketoglutarate35. Therefore, increased alpha ketoglutarate levels may also activate mTOR pathways and induce cellular hyperproliferation.\n\nAltered alpha ketoglutaric acid levels may also play a role within the hyperactive immunogenic state of psoriasis, in which lymphocytes, macrophages, and neutrophils secrete the inflammatory cytokines TNF-α, IL-1, and IL-6. In cell cultures, alpha ketoglutarate appears to enhance macrophage cytotoxicity by significantly increasing TNF-α and nitric oxide release36. Furthermore, TNF-α release from macrophages can activate collagen synthesis in dermal fibroblasts of psoriatic skin. Previous studies have observed that psoriasis is associated with increased collagen synthesis37–39. Collagen functions as an abundant connective tissue protein and provides structural integrity for the epidermis. During collagen synthesis, alpha ketoglutarate functions as a cofactor that activates Prolyl-4-Hydroxylase (P4H), an enzyme that mediates the post-translational modification of pre-procollagen peptides essential for formation of the collagen triple helix. Increased enzymatic activity of P4H enhances collagen synthesis in psoriatic lesions. Moreover, alpha ketoglutarate increases the available pool of proline residues required for collagen synthesis. Transamination of alpha ketoglutarate into glutamate, with subsequent conversion into proline, provides P4H with additional proline substrates to synthesize the hydroxyproline required in collagen formation37. Taken together, higher concentrations of alpha ketoglutaric acid may contribute to the structural properties as well as the immune and inflammatory properties of psoriasis.\n\nAlpha ketoglutaric acid. As noted earlier, alpha ketoglutarate can act to facilitate collagen synthesis in psoriasis patients. Although psoriasis patients exhibit elevated alpha ketoglutarate levels compared to controls, patients diagnosed with psoriasis and psoriatic arthritis had lower serum alpha ketoglutarate levels. This may be the result of a higher inflammatory burden experienced by these patients. Inflammatory cytokines, particularly interleukins and TNF-α, are up-regulated in the synovial fluid of psoriatic arthritis patients, further enhancing inflammatory joint destruction and periarticular bone loss40. Collagen functions as the primary structural component in cartilage, bones, and skin. Degradation of joint tissue in patients with psoriasis and psoriatic arthritis may significantly increase the demand for alpha ketoglutarate in an attempt to synthesize new collagen. This increased consumption may account for the decreased alpha ketoglutarate levels in patients with psoriasis and psoriatic arthritis.\n\nLignoceric acid. Lignoceric acid, a saturated very long-chain fatty acid (VLCA), is a minor fatty acid component found in human tissues and the bloodstream. In this study, serum lignoceric acid levels were elevated in patients with psoriasis with both skin and joint involvement when compared to patients with skin-limited psoriasis. The levels of saturated VLCFA in erythrocytes, specifically lignoceric acid, have been used to evaluate atherogenicity in patients with metabolic syndrome41. Similarly, Matsumori et al. (2013) found higher levels of lignoceric acid in the erythrocytes of patients with metabolic syndrome when compared to healthy controls41. This increase significantly correlated with specific atherogenic lipoprotein profiles and systemic inflammation in patients with metabolic syndrome41. A metabolic syndrome is characterized partly by insulin resistance resulting from chronically elevated levels of plasma free fatty acids that, when released into liver and muscle tissue, inhibit insulin secretion42. Epidemiologic studies have shown an increased prevalence of metabolic syndrome in patients with psoriatic arthritis compared to patients with psoriasis only43. As such, the increase in lignoceric acid levels is consistent with the notion that psoriasis patients afflicted with concomitant psoriatic arthritis may experience a greater inflammatory burden than patients with skin involvement alone.\n\nGlucuronic acid. Glucuronic acid is one of the main subunits that comprise the backbone of glycosaminoglycans (GAGs). In the present study, levels of glucuronic acid were increased in patients with psoriasis and psoriatic arthritis when compared to healthy controls, which corroborates the role of GAGs in relation to psoriasis. GAGs are complex, negatively-charged polysaccharides that bind with proteins to form proteoglycans. These structures are intimately involved in crucial components of cell signaling, cell adhesion and cell migration as they modulate the activity of the proteins that they bind44. Of note, GAGs have been proposed to store as well as activate growth factors in the extracellular matrix and cell surface44,45. This suggests that GAGs potentially play a significant role in influencing cell proliferation, differentiation, tissue remodeling, and regulation of extracellular matrix composition45–49. The GAGs present in the extracellular matrix of these cells may induce keratinocyte hyperproliferation and decrease epidermal differentiation in psoriasis.\n\nStudies have shown elevated levels of GAGs, specifically chondroitin sulfate and dermatan sulfate, in dermatologic and urine samples of patients with psoriasis. Chondroitin sulfate is a class of GAG that consists of a disaccharide backbone composed of alternating D-glucuronic acid and N-acetyl-D-galatosamine, whereas dermatan sulfate is synthesized from the epimerization of the glucuronic acid residues to iduronic acid at the polymer level50. Using antibody staining against chondroitin sulfate, Smetsers et al. (2004) found that the chondroitin sulfate was primarily expressed in the papillary dermis and the basal keratinocytes in normal skin samples, whereas there was a more diffuse staining extending into the reticular dermis in psoriatic skin samples51. In a study examining the localization of various GAGs on skin cells of normal and psoriatic skin, Saga et al. (1995) found that psoriatic epidermis exhibited increased chondroitin sulfate as well as dermatan sulfate, verified through digestion by enzymes specific to certain GAGs52. The researchers also noted a higher concentration of chondroitin sulfate and dermatan sulfate from the stratum basale to the lower stratum spinosum in psoriasis patients compared to patients with normal skin52. Moreover, Poulsen et al. (1983) reported that the concentration of GAGs increased by 28% and 62% in the dermal and urine samples, respectively, of psoriatic patients versus the healthy controls53. In another study, Poulsen et al. (1982) noted that the excretion of dermatan sulfate doubled to 104% (p < 0.01) and the excretion of chondroitin sulfate increased by 62% (p < 0.02) in psoriasis patients versus controls54. However, only the excretion of dermatan sulfate correlated with the fraction of body surface area involved in the psoriasis patients54. It has been suggested that the increased urinary excretion of GAGs indicates greater catabolism55.\n\nIn patients with psoriasis and psoriatic arthritis, chronic inflammation often leads to irreversible joint destruction56. Hyaluronan is another class of GAG composed of a D-glucuronic acid and N-acetyl-D-galatosamine backbone57. As a major component of the extracellular matrix in the articular cartilage, the joint destruction and degradation of hyaluronan into smaller oligosaccharides and its main components may account for the increase in glucuronic acid levels detected in our study. Although literature is scarce regarding the levels of hyaluronan in psoriatic arthritis, studies have reported an increase in serum hyaluronan in patients with rheumatoid arthritis when compared to healthy individuals58–61. Of note, hyaluronan has exhibited a diverse biological role with opposing dual functions in the inflammatory process57. Fragments of hyaluronan have been reported to enhance the inflammatory and catabolic response through the modulation of Toll-like receptor 2 signaling pathways62. While the suppression of hyaluronan synthesis alleviates inflammatory responses in some arthritic models, hyaluronan can act as an inflammatory activator as well as an inflammatory moderator63,64. Taken together, because glucuronic acid is a crucial backbone of GAGs, these results are consistent with our findings of elevated glucuronic acid in psoriatic serum samples.\n\nIn this study, a global metabolomics approach allowed an unbiased analysis of the entire pool of low molecular weight metabolites. The metabolomics data generated using this approach must be integrated with prior knowledge to identify specific metabolites for targeted analysis in future studies and to validate/confirm findings. Our study has several limitations. The small sample size may be associated with the inability to detect potential differences in certain metabolites. In this pilot study, while we captured PASI scores, psoriasis patients were allowed to maintain their usual treatment regimens. The effect of different treatment agents on metabolite perturbations could not be determined in this pilot study. Future studies with a larger patient cohort are necessary to confirm these findings.\n\nTo our knowledge, this is the first study to evaluate the comprehensive metabolome of patients with psoriasis. Differences in the serum metabolites of psoriasis patients, psoriasis patients with psoriatic arthritis, and control patients were detected through GC-TOF-MS, and further studies with larger sample sizes are necessary to confirm our findings. The development of a well-characterized metabolomics profile for patients with psoriasis and psoriatic arthritis will contribute to understanding pathophysiology of psoriasis and its associated comorbidities. The metabolite differences between psoriasis patients and healthy individuals detected by global metabolomics analysis help elucidate the underlying mechanisms of psoriasis and provide the foundations for therapeutic development.\n\n\nData availability\n\nF1000Research: Dataset 1. Data of metabolite differences in psoriasis and psoriatic arthritis, 10.5256/f1000research.4709.d3709065\n\n\nConsent\n\nAll the participants provided a written informed consent to publish the data reported in this study. This study was approved by the Institutional Review Board at University of California, Davis.", "appendix": "Author contributions\n\n\n\nAA and DG had full access to all of the data in the study and took responsibility for the integrity of the data and the accuracy of the data analysis. AA and MJ were responsible for the study concept and design. AA, DG, and OF analysed and interpreted the data. AA, JW, DG, BA, JD, OF drafted the manuscript. AA and DG provided critical revision of the manuscript for important intellectual content and statistical analysis. AA obtained funding for the study. AA performed the administrative, technical, material support, and study supervision.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work is supported by an Agency for Healthcare Quality and Research K08 Career Development Award 1K08HS018341-01 to AWA and a National Institutes of Health grant NIH U24 DK097154 to OF.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe would like to thank Sharon Cam for her contributions to the editing of the manuscript.\n\n\nReferences\n\nRachakonda TD, Schupp CW, Armstrong AW: Psoriasis prevalence among adults in the United States. J Am Acad Dermatol. 2014; 70(3): 512–6. PubMed Abstract \| Publisher Full Text\n\nGladman DD, Antoni C, Mease P, et al.: Psoriatic arthritis: epidemiology, clinical features, course, and outcome. Ann Rheum Dis. 2005; 64(Suppl 2): ii14–17. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMatsumori R, Miyazaki T, Shimada K, et al.: High levels of very long-chain saturated fatty acid in erythrocytes correlates with atherogenic lipoprotein profiles in subjects with metabolic syndrome. Diabetes Res Clin Pract. 2013; 99(1): 12–18. PubMed Abstract \| Publisher Full Text\n\nBoden G: Role of fatty acids in the pathogenesis of insulin resistance and NIDDM. Diabetes. 1997; 46(1): 3–10. PubMed Abstract \| Publisher Full Text\n\nSharma A, Gopalakrishnan D, Kumar R, et al.: Metabolic syndrome in psoriatic arthritis patients: a cross-sectional study. Int J Rheum Dis. 2013; 16(6): 667–673. PubMed Abstract \| Publisher Full Text\n\nTumova S, Woods A, Couchman JR: Heparan sulfate proteoglycans on the cell surface: versatile coordinators of cellular functions. Int J Biochem Cell Biol. 2000; 32(3): 269–288. PubMed Abstract \| Publisher Full Text\n\nZimmermann DR, Dours-Zimmermann MT, Schubert M, et al.: Versican is expressed in the proliferating zone in the epidermis and in association with the elastic network of the dermis. J Cell Biol. 1994; 124(5): 817–825. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoyd FT, Massagué J: Transforming growth factor-beta inhibition of epithelial cell proliferation linked to the expression of a 53-kDa membrane receptor. J Biol Chem. 1989; 264(4): 2272–2278. PubMed Abstract \| Publisher Full Text\n\nDamon DH, Lobb RR, D’Amore PA, et al.: Heparin potentiates the action of acidic fibroblast growth factor by prolonging its biological half-life. J Cell Physiol. 1989; 138(2): 221–226. PubMed Abstract \| Publisher Full Text\n\nRoberts R, Gallagher J, Spooncer E, et al.: Heparan sulphate bound growth factors: a mechanism for stromal cell mediated haemopoiesis. Nature. 1988; 332(6162): 376–378. PubMed Abstract \| Publisher Full Text\n\nThompson JA, Anderson KD, DiPietro JM, et al.: Site-directed neovessel formation in vivo. Science. 1988; 241(4871): 1349–1352. PubMed Abstract \| Publisher Full Text\n\nSilbert JE, Sugumaran G: Biosynthesis of chondroitin/dermatan sulfate. IUBMB life. 2002; 54(4): 177–186. PubMed Abstract \| Publisher Full Text\n\nSmetsers TF, van de Westerlo EM, ten Dam GB, et al.: Human single-chain antibodies reactive with native chondroitin sulfate detect chondroitin sulfate alterations in melanoma and psoriasis. J Invest Dermatol. 2004; 122(3): 707–716. PubMed Abstract \| Publisher Full Text\n\nSaga K, Takahashi M: Localization of anionic sites in normal and psoriatic epidermis: the effect of enzyme digestion on these anionic sites. Br J Dermatol. 1995; 132(5): 710–717. PubMed Abstract \| Publisher Full Text\n\nPoulsen JH, Cramers MK: The sulphate content of dermal and urinary glycosaminoglycans in psoriatics with increased excretion and increased dermal content of glycosaminoglycans. Scand J Clin Lab Invest. 1983; 43(3): 223–225. PubMed Abstract\n\nPoulsen JH, Cramers MK: Dermatan sulphate in urine reflects the extent of skin affection in psoriasis. Clin Chim Acta. 1982; 126(2): 119–126. PubMed Abstract \| Publisher Full Text\n\nPriestley GC: Urinary excretion of glycosaminoglycans in psoriasis. Arch Dermatol Res. 1988; 280(2): 77–82. PubMed Abstract \| Publisher Full Text\n\nAltomare G, Capsoni F: The diagnosis of early psoriatic arthritis. G Ital Dermatol Venereol. 2013; 148(5): 501–504. PubMed Abstract\n\nDicker KT, Gurski LA, Pradhan-Bhatt S, et al.: Hyaluronan: a simple polysaccharide with diverse biological functions. Acta Biomater. 2013; 10(4): 1558–70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEngstrom-Laurent A: Changes in hyaluronan concentration in tissues and body fluids in disease states. Ciba Found Symp. 1989; 143: 233–240; discussion 240–237, 281–235. PubMed Abstract \| Publisher Full Text\n\nSasaki Y, Uzuki M, Nohmi K, et al.: Quantitative measurement of serum hyaluronic acid molecular weight in rheumatoid arthritis patients and the role of hyaluronidase. Int J Rheum Dis. 2011; 14(4): 313–319. PubMed Abstract \| Publisher Full Text\n\nEngstrom-Laurent A, Hallgren R: Circulating hyaluronate in rheumatoid arthritis: relationship to inflammatory activity and the effect of corticosteroid therapy. Ann Rheum Dis. 1985; 44(2): 83–88. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaimela L, Heiskanen A, Kurki P, et al.: Serum hyaluronate level as a predictor of radiologic progression in early rheumatoid arthritis. Arthritis Rheum. 1991; 34(7): 815–821. PubMed Abstract \| Publisher Full Text\n\nQuero L, Klawitter M, Schmaus A, et al.: Hyaluronic acid fragments enhance the inflammatory and catabolic response in human intervertebral disc cells through modulation of toll-like receptor 2 signalling pathways. Arthritis Res Ther. 2013; 15(4): R94. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen WY, Abatangelo G: Functions of hyaluronan in wound repair. Wound Repair Regen. 1999; 7(2): 79–89. PubMed Abstract \| Publisher Full Text\n\nYoshioka Y, Kozawa E, Urakawa H, et al.: Suppression of hyaluronan synthesis alleviates inflammatory responses in murine arthritis and in human rheumatoid synovial fibroblasts. Arthritis Rheum. 2013; 65(5): 1160–1170. PubMed Abstract \| Publisher Full Text\n\nArmstrong AW, Wu J, Johnson MA, et al.: Data of metabolite differences in psoriasis and psoriatic arthritis. F1000Research. 2014. Data Source" }	[ { "id": "6483", "date": "10 Nov 2014", "name": "Trilokraj Tejasvi", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript is very well written and informative. The shortcomings of this study are highlighted in the discussion; the sample size, the usage of medications and the exploratory design. Although the statistical analyses used are appropriate, presenting nominal p values and the corrected p values would add reality to this study.", "responses": [] }, { "id": "6927", "date": "06 Jan 2015", "name": "J Lee", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nInteresting pilot study investigating metabolite differences between patients with psoriasis, those with psoriatic arthritis, and unaffected controls. I look forward to seeing these results replicated in a follow-up study which will include larger sample size. Controlling for treatment method, and comparison of patients who are on vs off treatment will also be necessary next steps. It will also be interesting to demonstrate in what manner such metabolites fluctuate with extent of disease. The mechanism behind these metabolite discrepancies and their significance in the disease pathogenesis also require further investigation. This study opens the door to multiple avenues of further study.", "responses": [] } ]	1	https://f1000research.com/articles/3-248
https://f1000research.com/articles/3-194/v1	13 Aug 14	{ "type": "Case Report", "title": "Case Report: Paroxysmal nocturnal hemoglobinuria in a woman heterozygous for G6PD A-", "authors": [ "Nieves Perdigones", "Mariela Morales", "Philip Mason", "Monica Bessler", "Nieves Perdigones", "Mariela Morales", "Monica Bessler" ], "abstract": "We describe a case of paroxysmal nocturnal hemoglobinuria (PNH) in a woman who is heterozygous for the glucose-6-phosphate dehydrogenase A-\n\n(G6PDA-) allele. PNH is associated with one or more clones of cells that lack complement inhibition due to loss of function somatic mutations in the PIGA gene. PIGA encodes the enzyme phosphatidylinositol glycan anchor biosynthesis, class A, which catalyses the first step of glycosylphosphatidylinisotol (GPI) anchor synthesis. Two GPI anchored red cell surface antigens regulate complement lysis. G6PD catalyses the first step of the pentose phosphate pathway and enzyme variants, frequent in some populations have been because they confer resistance to malaria, are associated with hemolysis in the presence of oxidizing agents including several drugs. The patient had suffered a hemolytic attack after taking Bactrim, a drug that precipitates hemolysis in G6PD deficient individuals. Since both G6PD and PIGA are X-linked we hypothesized that the PIGA mutation was on the X-chromosome carrying the G6PDA- allele. Investigations showed that in fact the PIGA mutation was on the X-chromosome carrying the normal G6PD B allele. We speculate that complement activation on G6PD A- red cells exposed to Bactrim might have triggered complement activation inducing the lysis of G6PD B PNH Type II red blood cells or that the patient may have had a PNH clone expressing G6PDA- at the time of the hemolytic episode.", "keywords": [ "In paroxysmal nocturnal hemoglobinuria (PNH) one or more clones of blood cells develops from stem cells that have an acquired mutation in the X-linked PIGA gene1. The PIGA gene encodes phosphatidylinositol glycan complementation class A", "an enzyme that catalyses an early and essential step in glycosylphosphatidylinositol (GPI) anchor synthesis. Thus cells are deficient in all GPI anchored proteins", "including CD55 and CD59 which regulate complement activation. PNH usually develops in patients with aplastic anemia (AA) and it is thought that PNH cells have a growth or survival advantage over the AA cells although the mechanism is not known2. PNH cells can be completely deficient in GPI anchored proteins (Type III) or partially deficient due to residual activity of the PIGA protein (Type II)", "while PNH Type I cells express GPI-linked proteins normally." ], "content": "Introduction\n\nIn paroxysmal nocturnal hemoglobinuria (PNH) one or more clones of blood cells develops from stem cells that have an acquired mutation in the X-linked PIGA gene1. The PIGA gene encodes phosphatidylinositol glycan complementation class A, an enzyme that catalyses an early and essential step in glycosylphosphatidylinositol (GPI) anchor synthesis. Thus cells are deficient in all GPI anchored proteins, including CD55 and CD59 which regulate complement activation. PNH usually develops in patients with aplastic anemia (AA) and it is thought that PNH cells have a growth or survival advantage over the AA cells although the mechanism is not known2. PNH cells can be completely deficient in GPI anchored proteins (Type III) or partially deficient due to residual activity of the PIGA protein (Type II), while PNH Type I cells express GPI-linked proteins normally.\n\nClinically, PNH is characterized by bone marrow failure, thrombosis and intravascular hemolysis. Recently the use of a complement inhibitor, eculizumab has greatly improved the quality of life of PNH patients as it causes a dramatic reduction in the hemolysis and thrombotic episodes, improvement in anemia, with a stabilization of the hemoglobin levels and reduced transfusion requirements3. Eculizumab leads to an increase in the number of circulating red blood cells that otherwise are subject to complement-mediated hemolysis4.\n\nGlucose-6-Phosphate Dehydrogenase (G6PD) deficiency is the most common red blood cell enzymopathy and is estimated to affect around 400 million people worldwide5. It is caused by mutations in the X-linked G6PD gene which usually lead to an unstable enzyme. G6PD is needed to maintain NADPH and consequently reduced glutathione levels in red blood cells. G6PD-deficient people, mainly males, can be asymptomatic but are subject to episodes of hemolysis when the red blood cells are subjected to oxidative stress caused by infections, certain drugs or in the case of favism, after eating fava beans6. Several polymorphic variants have been described with specific geographical distributions7. In the African population the most common deficient variant is the G6PD A- variant. Compared with normal G6PD, which is called G6PD B, G6PD A- has two amino acid substitutions Val68Met and Asn126Asp8. These are caused by mutations c.202 G->A and c.376A->G respectively. G6PD A- has a frequency of about 10% in Africans and African Americans. G6PD A differs from G6PD B only by the Asn126Asp change and is electrophoretically distinct but with no significant difference in activity. Though milder than other variants such as G6PD Mediterranean found in Italy, Greece and India, G6PD A- is associated with drug induced hemolysis and patients are advised against taking any substances from a list of those known to cause hemolysis. G6PD deficiency usually only affects hemizygous males and homozygous females but heterozygous females can be affected when, for example, biased X-inactivation has led to a predominance of red blood cells expressing the mutant protein9. Here we present a case of an African American woman who was heterozygous for G6PD deficiency and developed PNH, presenting an opportunity to observe the interaction of these two conditions.\n\n\nMaterials and methods\n\nPeripheral blood from patient CHOP277.01 was obtained after obtaining written informed consent according to the declaration of Helsinki. The Internal Review Board of the Hospital of the University of Pennsylvania approved this study. DNA and RNA were extracted by using QIAamp DNA and RNA Blood mini Kits, respectively, according to manufacturers’ instructions. Blood samples for fluorescent cytometry and electrophoretic analyses were obtained from EDTA tubes and experiments were performed within 2 hours of blood withdrawal.\n\nPCR primers to detect mutations confirming the G6PD A- genotype were designed with Primer3 v4.0. (primers for c.202 G->A mutation: forward 5’- agaagaagatctaccccaccatct-3’ and reverse 5’- ctggtacagagggcagaaccag-3’; primers for c.376A->G: forward 5’-catctgtctgtgtgtctgtctgtc-3’ and reverse 5’- ctcatagagtggtgggaggac-3’). Sanger sequencing was done by the Nucleic Acids core facility at CHOP.\n\nThe HUMARA assay was performed as previously described10. Briefly, HhaI digested and non digested DNA was subjected to PCR amplification of the first exon of the HUMARA locus (containing a CAG repeat) using fluorochrome-coupled primers. Amplification products were then migrated on an ABI PRISM 3100 Automatic Genetic Analyzer (Applied Biosystems). Allele calling and the area under the curve (AUC) were determined using GeneMapper v.4.0 software (Applied Biosystems). The AUC was used to calculate the skewing from X chromosome inactivation (XCI) The XCI ratio of the digested fraction was corrected with that of the undigested fraction to allow for preferential amplification of the smallest allele (i.e., the allele containing less CAG repeats). Skewing is present when the percentage of the predominant allele exceeds 74%. A percentage of predominant allele between 90% and 100% is considered extreme skewing.\n\nMeasurements of oxidative stress ROS assay was performed as previously described11. Briefly, red blood cells were incubated with 20-70-dichlorofluorescein diacetate (DCF; Sigma) dissolved in methanol. After incubation at 37ºC for 15 minutes in a humidified atmosphere of 5% CO2 in air, the cells were washed, resuspended in PBS and analyzed by flow cytometry (FACSCalibur; Becton-Dickinson, Immunofluorometry Systems, Mountain View, CA, USA). The mean fluorescence channel (MFC) was calculated by FACSDiva software. The identity of the red cell population was verified by staining with an antibody anti to glycophorin-A. To determine the presence of GPI proteins, cells were labeled with a phycoerythrin-conjugated anti-CD55 antibody. For our experiment, cells from a non PNH- non G6PD individual served as control. The MFC of cells stained with DCF, is proportional to generation of ROS.\n\nThe electrophoretic mobility of the protein was performed in cellogel strips as previously described12. Hemolysates treated with and without acidified serum were run in order to assess differences in mobility of the G6PD enzyme.\n\n\nCase report\n\nThe patient, a 23 year old African American woman, presented with left upper abdominal pain, vomiting and blurry vision. She had a four year history of episodes of hemolysis, abdominal pain and dark urine.\n\nShe was diagnosed with acute hemolytic exacerbation of PNH and was admitted. The patient was found to have anemia, leukocytosis and mild transaminitis. A Magnetic Resonance Venogram (MRV) of the abdomen and pelvis revealed several ill-defined low attenuation lesions of the posterior segment of the right hepatic lobe consistent with liver thrombosis so she was started on anticoagulation a concomitant urinary tract infection was treated with Trimethoprim-Sulfamethoxazole (Bactrim) and she was discharged.\n\nAfter discharge, she followed up with a hematologist. On review of systems, the patient complained of a hemolytic attack with dark urine after taking Bactrim. On physical exam, she was found to have mildly icteric sclera. On further questioning of family history of other hematologic disorders, the patient said that one of her nephews has Glucose-6-Phosphate-Dehydrogenase deficiency. Because of her diagnosis of PNH and abdominal thrombosis, she was started on Eculizumab. The laboratory workup revealed a white blood cell count 4.6 k/uL, RBC count of 3.8 k/uL, hemoglobin of 11.8 g/dL, hematocrit 35%, MCV 91 fl, platelets 327 k/uL, reticulocytes 24.5%, PTT 27.8, INR 1.7, D dimer 2.5 ug/mL, bilirubin 1.3 mg/dL, bilirubin direct 0.2 mg/dL, bilirubin indirect 1.1 mg/dL, ANC 2.7 k/uL, ALC 1.3 k/uL, G6PD screen normal, LDH 720 U/L. The flow cytometry at this point revealed: 69% of the red blood cells were partially deficient for CD59 and 9.1% of the red blood cells were completely deficient for CD59; 86.3% of the granulocytes were partially deficient for CD59 and 6.4% were completely deficient for CD59.\n\n\nResults\n\nThe family history and the case history suggested that the patient may have both G6PD deficiency and PNH, since the G6PD deficiency might explain the hemolysis precipitated by Bactrim, a drug that is reported to cause hemolysis in G6PD patients. Sequencing of DNA from her granulocytes confirmed that she was heterozygous for G6PD A- having a G6PD B allele on one X-chromosome and a G6PD A- allele on the other. This finding raised the question as to whether the somatic PIGA mutation causing her PNH took place on the X-chromosome carrying the B or the A- G6PD gene. The flow cytometry data showed that the patient most likely had 2 PNH clones, a class I clone (partial deficiency) of about 86% and a class II clone (complete deficiency) of about 6%. The HUMARA assay, which measures X-inactivation, showed a single clone of about 90% (Figure 1), suggesting that in both clones the mutation had taken place on the same X-chromosome. We hypothesized that the mutations in PIGA would have taken place on the chromosome carrying the G6PD A- allele since this would help explain the patient’s reaction to Bactrim. Another factor was that treatment with eculizimab, by inhibiting lysis of PNH red cells may have led to a higher level of PNH (and concomitantly G6PD deficient) red cells than would be present in untreated patients. To determine which G6PD allele was expressed in the PNH clone we sequenced cDNA from the patient’s granulocytes. The sequencing trace showed that the vast majority of expressed G6PD cDNA contained the wild type (G6PD B) sequence at both nucleotides where it differs from G6PD A- (Figure 2), leaving us to conclude that the PIGA mutations had taken place on the X-chromosome containing the G6PD B allele. This was confirmed at the protein level since the red blood cells lysed by acidified serum (the PNH cells) contained most of the G6PD activity while the residual cells did not contain detectable G6PD activity. While developing our hypothesis, which turned out to be incorrect, we also considered whether PNH/G6PD A- cells might have high levels of oxidative stress since both G6PD deficiency and PNH have been shown to be associated with elevated levels of reactive oxygen species11,13. We found that the patient’s red blood cells contained ROS levels that were significantly higher than those from healthy controls, though surprisingly we did not detect any difference in ROS between PNH (CD55-) and normal (CD55+) cells (Figure 3).\n\nThe panel at the bottom shows the migration of the 2 microsatellite alleles in patient CHOP277.01 revealed by PCR analysis of a region of the Androgen receptor gene on the X-chromosome. The amplified region contains both a polymorphic repeat and a site for the methylation sensitive restriction enzyme HhaI. This site is methylated on the inactive X-chromosome. The top panel shows the same sample digested with HhaI before the PCR so only the fragment on the inactive X-chromosome is amplified. An imbalance in the allelic ratio reflects an imbalance in X inactivation and therefore indicates clonality.\n\nA1 and A2 shows sequencing of genomic DNA around the 202 G->A and the 376 A->G mutation that lead to Val68Met and Asn126Asp changes in G6PD, respectively. Figures B1 and B2 show the cDNA sequence of the exact same mutations in the peripheral blood of the patient. The WT (B) allele is expressed in the majority of cells.\n\nPeripheral blood mononuclear cells from the patient and a normal control were treated with the oxidation sensitive dye, CM-H2DCFDA, and the conversion to its oxidized fluorescent derivative assessed by flow cytometry. The fluorescence distribution histogram and the mean fluorescence channels (MFC) of each population derived from the normal control (orange) and the patient (blue for CD55 negative and red for CD55 positive cells) are shown.\n\n\nDiscussion\n\nPNH is a rare condition, having an incidence of about 1 in a million, so the co-incidental finding of a female with PNH and heterozygous for G6PD deficiency was an opportunity to observe the interaction between these 2 conditions which both involve red blood cell hemolysis mediated by X-linked genes. Notably the first demonstration that PNH was a clonal disease took advantage of a female PNH patient who was heterozygous for the electrophoretic variant G6PD A14. In this patient both isozymes were present in a lysate of total red blood cells, but only one was present after acidified serum lysis, demonstrating clonality of the PNH cells.\n\nIn the case discussed here a female African American patient with PNH suffered episodes of hemolysis, often following treatment with Trimethoprim-Sulfamethoxazole (Bactrim), one of the drugs that is known to cause hemolysis in G6PD patients15. When it emerged that she was heterozygous for G6PD A- we hypothesized that her expanded PNH clones may be expressing only the G6PD A- protein. The hypothesis proved incorrect and the clone expressed the wild type G6PD allele. A possible explanation is that complement activation on G6PD A- red cells exposed to Bactrim might have triggered complement activation inducing the lysis of G6PD B PNH Type II red blood cells16. Alternatively since PNH patients often have several clones17 which change in prevalence it is possible that earlier she did have a G6PD A- clone but this was replaced by wild type clones. Finally we can also speculate that the combination of G6PD and PIGA deficiency confers a serious growth disadvantage and PNH clones in this situation are more likely to be G6PD wild type. There is no clear mechanism for this however as G6PD A- nucleated cells have similar enzyme activity to WT cells18 – the deficiency becoming apparent in red blood cells, which do not synthesize new protein19.\n\n\nPatient consent\n\nWritten informed consent for publication of their clinical details was obtained from the patient.", "appendix": "Author contributions\n\n\n\nPJM and MB conceived the study, NP designed the experiments and carried out the research. MM collected and collated clinical data. All authors contributed to preparing a draft of the manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe work has been supported by the Buck Family Endowed Chair in Hematology, and by NCI NIH grants 2R01CA106995 to PJ Mason, and 2R01 CA105312 to M Bessler.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nParker CJ: Paroxysmal nocturnal hemoglobinuria. Curr Opin Hematol. 2012; 19(3): 141–8. PubMed Abstract \| Publisher Full Text\n\nLuzzatto L, Bessler M, Rotoli B: Somatic mutations in paroxysmal nocturnal hemoglobinuria: a blessing in disguise? Cell. 1997; 88(1): 1–4. PubMed Abstract \| Publisher Full Text\n\nMcKeage K: Eculizumab: a review of its use in paroxysmal nocturnal haemoglobinuria. Drugs. 2011; 71(17): 2327–45. PubMed Abstract \| Publisher Full Text\n\nKelly RJ, Hill A, Arnold LM, et al.: Long-term treatment with eculizumab in paroxysmal nocturnal hemoglobinuria: sustained efficacy and improved survival. Blood. 2011; 117(25): 6786–92. PubMed Abstract \| Publisher Full Text\n\nMason PJ, Bautista JM, Gilsanz F: G6PD deficiency: the genotype-phenotype association. Blood Rev. 2007; 21(5): 267–83. PubMed Abstract \| Publisher Full Text\n\nBeutler E: G6PD deficiency. Blood. 1994; 84(11): 3613–36. PubMed Abstract\n\nBeutler E: G6PD: population genetics and clinical manifestations. Blood Rev. 1996; 10(1): 45–52. PubMed Abstract \| Publisher Full Text\n\nHirono A, Beutler E: Molecular cloning and nucleotide sequence of cDNA for human glucose-6-phosphate dehydrogenase variant A(-). Proc Natl Acad Sci U S A. 1988; 85(11): 3951–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAu WY, Lam V, Pang A, et al.: Glucose-6-phosphate dehydrogenase deficiency in female octogenarians, nanogenarians, and centenarians. J Gerontol A Biol Sci Med Sci. 2006; 61(10): 1086–9. PubMed Abstract \| Publisher Full Text\n\nAllen RC, Zoghbi HY, Moseley AB, et al.: Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation. Am J Hum Genet. 1992; 51(6): 1229–39. PubMed Abstract \| Free Full Text\n\nAmer J, Zelig O, Fibach E: Oxidative status of red blood cells, neutrophils, and platelets in paroxysmal nocturnal hemoglobinuria. Exp Hematol. 2008; 36(4): 369–77. PubMed Abstract \| Publisher Full Text\n\nRattazzi MC, Corash LM, van Zanen GE, et al.: G6PD deficiency and chronic hemolysis: four new mutants--relationships between clinical syndrome and enzyme kinetics. Blood. 1971; 38(2): 205–18. PubMed Abstract\n\nRonquist G, Theodorsson E: Inherited, non-spherocytic haemolysis due to deficiency of glucose-6-phosphate dehydrogenase. Scand J Clin Lab Invest. 2007; 67(1): 105–11. PubMed Abstract \| Publisher Full Text\n\nOni SB, Osunkoya BO, Luzzatto L: Paroxysmal nocturnal hemoglobinuria: evidence for monoclonal origin of abnormal red cells. Blood. 1970; 36(2): 145–52. PubMed Abstract\n\nSerpa JA, Villarreal-Williams E, Giordano TP: Prevalence of G6PD deficiency in a large cohort of HIV-infected patients. J Infect. 2010; 61(5): 399–402. PubMed Abstract \| Publisher Full Text\n\nArese P, Gallo V, Pantaleo A, et al.: Life and Death of Glucose-6-Phosphate Dehydrogenase (G6PD) Deficient Erythrocytes - Role of Redox Stress and Band 3 Modifications. Transfus Med Hemother. 2012; 39(5): 328–334. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMortazavi Y, Merk B, McIntosh J, et al.: The spectrum of PIG-A gene mutations in aplastic anemia/paroxysmal nocturnal hemoglobinuria (AA/PNH): a high incidence of multiple mutations and evidence of a mutational hot spot. Blood. 2003; 101(7): 2833–41. PubMed Abstract \| Publisher Full Text\n\nMarks PA, Gross RT: Erythrocyte glucose-6-phosphate dehydrogenase deficiency: evidence of differences between Negroes and Caucasians with respect to this genetically determined trait. J Clin Invest. 1959; 38(12): 2253–62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMason PJ: New insights into G6PD deficiency. Br J Haematol. 1996; 94(4): 585–91. PubMed Abstract" }	[ { "id": "5816", "date": "28 Aug 2014", "name": "Rosario Notaro", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPerdigones and collaborators report an interesting clinical case about the association of PNH and G6PD deficiency. However, the timing of the clinical history is not clear and the report is scanty of some relevant clinical/laboratory data.Major scientific points.The Authors should provide the exact timing of entire clinical history: diagnosis; infective episode resulting in the prescription of trimethoprim-sulfamethoxazole -co-trimoxazole- (which dose?); start and stop of co-trimoxazole; “dark urine episode” associated with co-trimoxazole; start of eculizumab; time of biological studies. It is extremely important that the Authors provide more details about the timing and the features of the hemolytic attack apparently associated with co-trimoxazole: how long after the start of co-trimoxazole treatment the patient experienced the “dark urine episode”? There are any objective data at the time of this “dark urine episode” or the episode has been just self-reported? At the time of this “dark urine episode” there were any signs/symptoms of an ongoing infective condition? The Authors provide clinical/laboratory details only at one time point, that seems be after the start of eculizumab (how long after?). They should provide such clinical/laboratory details at time of diagnosis, at the time of the co-trimoxazole associated “hemolytic attack”, at start of eculizumab treatment, etc.: blood count, absolute reticulocyte count, LDH levels (providing the normal range), flow citometry, etc. In PNH patients the presence of red blood cells with partial deficiency of GPI-linked molecules is relatively common. However, the presence of granulocyte/monocyte with partial deficiency of GPI-linked molecules is uncommon: thus, it would be interesting to show the dot plot of the “CD59/lineage marker” analysis of granulocytes and monocytes of this patient. The Authors, at variance with their starting hypothesis, have clearly proven that in this patient PNH cells express the wild type G6PD B. They provide 2 possible explanations for the co-trimoxazole-associated “hemolytic crisis” observed in this patient. These hypotheses are interesting but their probability is very low. I suggest that the Authors should discuss a much more likely explanation: this “hemolytic crisis” was just due to the infective condition that led to the prescription of co-trimoxazole.Minor pointsIn the Introduction the Authors report the classical classification of PNH cells as Type III (completely deficient in GPI anchored proteins), Type II (partially deficient) and Type I (normal display of GPI-linked proteins). However, in the Results they write about “class I clone (partial deficiency) … and class II clone (complete deficiency) “: this is extremely confusing. In the Results the 2 sentences (from “We hypothesized that …” to “…red cells than would be present in untreated patients.”) seem to suggest that the relative expansion of the “PNH (and concomitantly G6PD deficient) red cells” following eculizumab treatment could have played a role in the co-trimoxazole associated hemolytic attack. The Authors should rephrase these sentences since eculizumab has been started after the co-trimoxazole associated hemolytic attack. Trimethoprim-sulfamethoxazole (co-trimoxazole) is a generic drug name thus it should not be capitalized. In the Results, “eculizumab” should not be capitalized. The brand name Bactrim should be replaced with the generic drug name. The levels of ROS in the patient red cells should be compared with a group of healthy controls: the comparison with only one control does not allow any conclusion. The Authors should report the concentration of DCF used for ROS detection.", "responses": [ { "c_id": "1028", "date": "15 Oct 2014", "name": "Philip Mason", "role": "Author Response F1000Research Advisory Board Member", "response": "Perdigones and collaborators report an interesting clinical case about the association of PNH and G6PD deficiency. However, the timing of the clinical history is not clear and the report is scanty of some relevant clinical/laboratory data.Major scientific points.The Authors should provide the exact timing of entire clinical history: diagnosis; infective episode resulting in the prescription of trimethoprim-sulfamethoxazole -co-trimoxazole- (which dose?); start and stop of co-trimoxazole; “dark urine episode” associated with co-trimoxazole; start of eculizumab; time of biological studies.In our revised manuscript we include a fuller, more detailed case history. Of note is the fact that the patient presented to the specialty clinic with a probably 6 year history of PNH and that most of the patients past history relies on records and notes. Here we demonstrate that the patient has indeed both conditions PNH and G6PD deficiency. With these results in hand we try to explain the patient’s history and clinical observations. It was not our intention to prove that indeed the hemolysis described by the emergency physician after co-trimoxazole was indeed triggered by the drug, but rather to evaluate and discuss the possibility as the patient carries on her chart the diagnosis of co-trimoxazole hypersensitity due to this incident. We try to make this clearer in the case description. We hope this will answer the criticism of the reviewer. It is extremely important that the Authors provide more details about the timing and the features of the hemolytic attack apparently associated with co-trimoxazole: how long after the start of co-trimoxazoletreatment the patient experienced the “dark urine episode”? There are any objective data at the time of this “dark urine episode” or the episode has been just self-reported? At the time of this “dark urine episode” there were any signs/symptoms of an ongoing infective condition?The discussion above and the revised version provides many more details. The Authors provide clinical/laboratory details only at one time point, that seems be after the start of eculizumab (how long after?). They should provide such clinical/laboratory details at time of diagnosis, at the time of the co-trimoxazole associated “hemolytic attack”, at start of eculizumab treatment, etc.: blood count, absolute reticulocyte count, LDH levels (providing the normal range), flow citometry, etc.We have provided all the history we can obtain in the revised version. In PNH patients the presence of red blood cells with partial deficiency of GPI-linked molecules is relatively common. However, the presence of granulocyte/monocyte with partial deficiency of GPI-linked molecules is uncommon: thus, it would be interesting to show the dot plot of the “CD59/lineage marker” analysis of granulocytes and monocytes of this patient.The diagnosis of PNH and the subtype analysis of PNH granulocytes was performed in a CLIA approved clinical laboratory, and their test results are reported here. The individual dot blots are not available to us. However the senior author who was involved in the setup and quality assessment of flow-cytometric PNH testing at this institution fully trusts their analysis. We agree with the reviewer that partial GPI-anchor deficiency in granulocytes is less frequently observed and very much depends on the underlying mutation and the antibody chosen for the analysis. The Authors, at variance with their starting hypothesis, have clearly proven that in this patient PNH cells express the wild type G6PD B. They provide 2 possible explanations for the co-trimoxazole-associated “hemolytic crisis” observed in this patient. These hypotheses are interesting but their probability is very low. I suggest that the Authors should discuss a much more likely explanation: this “hemolytic crisis” was just due to the infective condition that led to the prescription of co-trimoxazole.We agree that this is a possible alternative explanation and have added this in the discussion however considering the rather bland urine sediment we actually favor the other two possible explanations.Minor pointsIn the Introduction the Authors report the classical classification of PNH cells as Type III (completely deficient in GPI anchored proteins), Type II (partially deficient) and Type I (normal display of GPI-linked proteins). However, in the Results they write about “class I clone (partial deficiency) … and class II clone (complete deficiency) “: this is extremely confusing.This has been corrected In the Results the 2 sentences (from “We hypothesized that …” to “…red cells than would be present in untreated patients.”) seem to suggest that the relative expansion of the “PNH (and concomitantly G6PD deficient) red cells” following eculizumab treatment could have played a role in the co-trimoxazole associated hemolytic attack. The Authors should rephrase these sentences since eculizumab has been started after the co-trimoxazole associated hemolytic attack.The timing of the eculizimab treatment and the hemolysis are given in the detailed case report that we now provide. Trimethoprim-sulfamethoxazole (co-trimoxazole) is a generic drug name thus it should not be capitalized. In the Results, “eculizumab” should not be capitalized.We have corrected this. The brand name Bactrim should be replaced with the generic drug name.We have corrected this. The levels of ROS in the patient red cells should be compared with a group of healthy controls: the comparison with only one control does not allow any conclusion.The experiment was carried out with several controls and it confirms the results of Amer et al., cited as our ref 14 that ROS are elevated in PNH. Our point is a small one, that we might expect a further elevation if we compare PNH and normal cells from this patient, if indeed PIGA- is linked with G6PDA-. We didn’t find this. We include the sentence “The figure shows a representative example of 4 normal controls that gave similar results” to clarify this. The Authors should report the concentration of DCF used for ROS detection.We have done this." } ] }, { "id": "6022", "date": "04 Sep 2014", "name": "ANASTASIOS KARADIMITRIS", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPerdigones and colleagues describe the rare co-occurence in the same patient of two X-linked disorders, one acquired the other inherited, both causing intravascular haemolysis. However how the interaction of the two disorders led to the clinical episode described in the case report is not clear because the temporal analysis of the clinical events and the associated laboratory tests are not presented in sufficient detail.Charting clinical events, labs and therapeutic interventions might make association of the heamolytic episodes with co-trimoxazole or intercurrent infection clearer. It appears that the majority of the PNH clone (in both granulocytes and red cells) is type II, i.e., only partially deficient of GPI. One assumes that this picture was obtained after the haemolytic attack. If so, it could be that type III, i.e., severely deficient RBC were indeed G6PD-deficient. Was flow-cytometry performed after Eculizumab treatment? Eculizumab would protect type III RBC from lysis and thus would allow re-assessment of G6PD activity. Minor pointsWas anti-CD55 or –CD59 was used for flow analysis? The authors state anti-CD55 in methods but describe anti-CD59 in results. Normal ranges of lab tests need to be provided ‘Another factor was that treatment with eculizumab, by inhibiting lysis of PNH red cells may have led to a higher level of PNH (and concomitantly G6PD deficient) red cells than would be present in untreated patients.’ This statement is rather irrelevant because eculizumab was started after treatment with co-trimoxazole. Need to use Greek characters appropriately, i.e., ul should be ml", "responses": [ { "c_id": "1029", "date": "15 Oct 2014", "name": "Philip Mason", "role": "Author Response F1000Research Advisory Board Member", "response": "It appears that the majority of the PNH clone (in both granulocytes and red cells) is type II, i.e., only partially deficient of GPI. One assumes that this picture was obtained after the haemolytic attack. If so, it could be that type III, i.e., severely deficient RBC were indeed G6PD-deficient. Was flow-cytometry performed after Eculizumab treatment? Eculizumab would protect type III RBC from lysis and thus would allow re-assessment of G6PD activity.We agree with this comment. Of course it is possible that at the time of the observation of association of co-trimoxazole associated hemolysis a different PNH clone was more prevalent than when analyzed for G6PD deficiency. We included this possibility in our discussion. The most recent flow cytometry was performed when the patient was on eculizimab. Minor pointsWas anti-CD55 or –CD59 was used for flow analysis? The authors state anti-CD55 in methods but describe anti-CD59 in results. The diagnosis for PNH was performed in a CLIA approved clinical laboratory of the hospital.We present the results for CD59. CD55 was also tested however the discrimination between the three populations is more difficult. Normal ranges of lab tests need to be provided.We have included normal ranges for laboratory tests in parentheses after the patient’s values. Another factor was that treatment with eculizumab, by inhibiting lysis of PNH red cells may have led to a higher level of PNH (and concomitantly G6PD deficient) red cells than would be present in untreated patients.’ This statement is rather irrelevant because eculizumab was started after treatment with co-trimoxazole.We think this statement is valid because treatment with eculizimab increases the level of PIGA deficient red cells. On our hypothesis that the G6PDA- allele was linked with PIGA- then PIGA-,G6PDA- red cells would increase. Our hypothesis however was not correct. Need to use Greek characters appropriately, i.e., ul should be ml We have corrected our incorrect use of Greek characters." } ] } ]	1	https://f1000research.com/articles/3-194
https://f1000research.com/articles/3-95/v1	24 Apr 14	{ "type": "Web Tool", "title": "shRNA-seq data analysis with edgeR", "authors": [ "Zhiyin Dai", "Julie M. Sheridan", "Linden J. Gearing", "Darcy L. Moore", "Shian Su", "Ross A. Dickins", "Marnie E. Blewitt", "Matthew E. Ritchie", "Zhiyin Dai", "Julie M. Sheridan", "Linden J. Gearing", "Darcy L. Moore", "Shian Su", "Ross A. Dickins", "Marnie E. Blewitt" ], "abstract": "Pooled short hairpin RNA sequencing (shRNA-seq) screens are becoming increasingly popular in functional genomics research, and there is a need to establish optimal analysis tools to handle such data. Our open-source shRNA processing pipeline in edgeR provides a complete analysis solution for shRNA-seq screen data, that begins with the raw sequence reads and ends with a ranked lists of candidate shRNAs for downstream biological validation. We first summarize the raw data contained in a fastq file into a matrix of counts (samples in the columns, hairpins in the rows) with options for allowing mismatches and small shifts in hairpin position. Diagnostic plots, normalization and differential representation analysis can then be performed using established methods to prioritize results in a statistically rigorous way, with the choice of either the classic exact testing methodology or a generalized linear modelling that can handle complex experimental designs. A detailed users’ guide that demonstrates how to analyze screen data in edgeR along with a point-and-click implementation of this workflow in Galaxy are also provided. The edgeR package is freely available from http://www.bioconductor.org.", "keywords": [ "Pooled short hairpin RNA sequencing (shRNA-seq) screens couple RNA interference (RNAi) with second generation sequencing to enable researchers to elucidate gene function in an unbiased", "high-throughput manner1. Several recent high impact studies have exploited this technology to discover novel genes involved in processes including cell fate decisions of normal and cancer cells", "and to generate genetic interaction maps in mammalian cells2–4." ], "content": "Introduction\n\nPooled short hairpin RNA sequencing (shRNA-seq) screens couple RNA interference (RNAi) with second generation sequencing to enable researchers to elucidate gene function in an unbiased, high-throughput manner1. Several recent high impact studies have exploited this technology to discover novel genes involved in processes including cell fate decisions of normal and cancer cells, and to generate genetic interaction maps in mammalian cells2–4.\n\nPooled shRNA screening relies on the stable genomic integration (often by viral transduction) of expression cassettes that allow stable or inducible expression of shRNAs targeting specific genes in a large population of cells. Depending on the biological question of interest, typically two or more cell populations are compared either in the presence or absence of a selective pressure, or as a time-course before and after a selective pressure is applied. Gain of shRNA representation within a pool suggests that target gene knockdown confers some sort of advantage to a cell. Similarly, genes whose knockdown is disadvantageous may be identified through loss of shRNA representation. Screening requires a library of shRNA constructs in a lentiviral or retroviral vector backbone that is used to generate a pool of virus for transducing cells of interest. The relative abundance of these shRNAs in transduced cells is then quantified by PCR amplification of proviral integrants from genomic DNA using primers designed to amplify all shRNA cassettes equally, followed by second-generation amplicon sequencing (Figure 1A). Sample-specific primer indexing allows many different conditions to be analyzed in parallel.\n\n(A) Structure of the amplicons sequenced in a typical shRNA-seq screen. Each one contains sample and hairpin specific sequences at predetermined locations. After sequencing, the raw data is available in a fastq file. (B) The main steps and functions used in an analysis of shRNA-seq screen data in edgeR are shown. (C) Example of a multidimensional scaling (MDS) plot showing the relationships between replicate dimethyl sulfoxide (DMSO) and Nutlin treated samples (data from Sullivan et al. (2012)3). MDS plots provide a quick display of overall variability in the screen and can highlight inconsistent samples. (D) Plot of log2-fold-change versus hairpin abundance (log2CPM) for the same data. Hairpins with a false discovery rate < 0.05 from an exact test analysis in edgeR (highlighted in red) may be prioritized for further validation.\n\nAs the popularity of this approach grows, there is a need to develop suitable analysis pipelines to handle the large volumes of raw data that each screen generates. The major steps in an analysis involve processing the raw sequence reads, assessing the data quality and determining representational differences in the screen in a statistically rigorous way.\n\nTwo pipelines are currently available for this task. The shALIGN program5 is a custom Perl script that trims the sequence reads to the pre-defined base positions and then matches these to a library of hairpin sequences. Mismatch bases are permitted, and any ambiguous matches are ignored from the final hairpin count. Statistical analysis of the data is then performed using the shRNAseq R package5, which calculates log-ratios of the counts from each screen replicate, normalizes these values and ranks hairpins by their median, mean or t-statistic. Another solution is the BiNGS!SL-seq program6 that uses Bowtie to perform sequence mapping followed by statistical analysis in edgeR7.\n\nIn this article, we describe a complete analysis solution for shRNA-seq screens accessible from within the edgeR package available from Bioconductor8.\n\n\nImplementation\n\nA summary of the main steps in a typical shRNA-seq analysis alongside the functions in edgeR that perform each task is given in Figure 1B.\n\nOur sequence counting procedure has been tailored for screens where PCR amplified shRNA constructs of known structure are sequenced using second generation sequencing technology (Figure 1A). The location of each index and hairpin sequence is used to determine matches between a specified list of index and hairpin sequences expected in the screen with the sequences in the fastq file. Mismatches in the hairpin sequence are allowed to accommodate sequencing errors, as are small shifts in the position of the hairpin sequence within the read. Analysis of unpublished in-house data reveals that allowing for mismatches can yield up to 4.4% additional reads, and shifting an extra 2.6%. This simple searching strategy is implemented in C, with the user interface provided by the processHairpinReads function in edgeR. Input to this function consists of a fastq file/s, a second file containing sample IDs and their index sequences and a third file listing hairpin IDs and their sequences (the latter files are tab-delimited). A screen with 100 million reads (one lane from an Illumina HiSeq 2000) can be processed in 2-15 minutes depending on the processing parameters. Fastq processing requires minimal RAM, allowing analysis to be completed on any standard computer with R9 installed.\n\nThe matrix of counts returned by the processHairpinReads function, which contains hairpins in the rows and samples in the columns, is stored as a DGEList object so that it is fully interoperable with the downstream analysis options available in edgeR. Such an object can also be created directly by the user in the event that the hairpin counts have been summarized by alternate means.\n\nNext, the data quality of a screen can be assessed conveniently using multidimensional scaling (MDS) plots via plotMDS (Figure 1C) and access to a range of normalization options is available through the calcNormFactors function.\n\nThe shRNAseq software5 assumes simple experimental set-ups (e.g. comparing two conditions) that are unsuitable in more complicated situations, such as time-course designs. In edgeR, screens can be analyzed using either the classic method10, ideal for simple two-group comparisons, or generalized linear models (GLMs)11 for more complex screens with multiple conditions (using the glmFit function). This framework can accommodate hairpin-specific variation of both a technical and biological nature as estimated via the estimateDisp function and visualized using plotBCV, which plots biological variability as a function of average hairpin abundance. Statistical testing for changes in shRNA abundance between conditions of interest (typically over time) is carried out using exact (see exactTest function) or likelihood ratio (glmLRT) tests that allow results to be ranked by significance using the topTags function and plotted using the plotSmear function (Figure 1D).\n\nGene set analysis tools available via roast12 and camera13 allow researchers to further test and prioritize screen results. This capability can be used to obtain a gene-by-gene ranking, rather than a hairpin-specific one, which can be helpful when shRNA libraries contain multiple hairpins targeting each gene.\n\nWe provide example data sets and a complete analysis script that demonstrate how to use the edgeR package to prioritize data from four different pooled shRNA-seq screens14. These examples were chosen to showcase edgeR’s ability to deal with experiments of varying size (from tens to thousands of hairpins) and complexity, from two-group situations, to settings with four groups, or a time-course design, where a GLM with a slope and intercept term is most appropriate. We have also developed a Galaxy tool15–17 that implements this workflow as a point-and-click application to improve accessibility for researchers who are unfamiliar with the R programming environment (Figure 2).\n\n(A) From the main screen, the user selects the appropriate input files and analysis options. (B) The results of an analysis are summarized in an HTML page that includes various diagnostic plots. (C) Output also includes a table of ranked results at the hairpin and gene-level (where appropriate) as well as barcode plots (D) that highlight the ranks of hairpins targeting a specific gene relative to all other hairpins in the data set.\n\n\nDiscussion\n\nAlthough the major functionality of edgeR has been developed with RNA-seq data in mind, the analysis of numerous in-house data sets14 and the results of others3 have demonstrated its utility for count data derived from shRNA-seq screens. edgeR provides users with a unique tool for the analysis of data from this emerging application of second generation sequencing technology, that is capable of handling both the biological variability and experimental complexity inherent in these screens. Provision of a Galaxy module puts these powerful statistical methods within reach of experimentalists. Future work will be focused on the use of a suitable control data set to compare this analysis pipeline with other approaches such as shRNAseq5. We anticipate that the approach for differential representation analysis described in this paper will also be useful in the analysis of short-guided RNA-seq (sgRNA-seq) knockout screens as facilitated by the clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) system18,19.\n\n\nSoftware availability\n\nedgeR is an R9 package distributed as part of the Bioconductor project8 (http://www.bioconductor.org). The Galaxy tool that implements this workflow is available from http://toolshed.g2.bx.psu.edu/view/shians/shrnaseq.", "appendix": "Author contributions\n\n\n\nZD and MER developed the hairpin counting software and SS developed the Galaxy tool. JMS, LJG, DLM and MEB generated the screen data analyzed in the user guide that accompanies this article and RAD developed the hairpin technology. All authors wrote and approved the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was supported by NHMRC Project grants 1050661 (MER) and 1059622 (MER and MEB), Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank Matthew Wakefield and Gordon Smyth for advice on data analysis, Cynthia Liu for code testing and our many collaborators at the WEHI whose research has motivated this work.\n\n\nReferences\n\nBassik MC, Lebbink RJ, Churchman LS, et al.: Rapid creation and quantitative monitoring of high coverage shRNA libraries. Nat Methods. 2009; 6(6): 443–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZuber J, Shi J, Wang E, et al.: RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia. Nature. 2011; 478(7370): 524–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSullivan KD, Padilla-Just N, Henry RE, et al.: ATM and MET kinases are synthetic lethal with nongenotoxic activation of p53. Nat Chem Biol. 2012; 8(7): 646–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBassik MC, Kampmann M, Lebbink RJ, et al.: A systematic mammalian genetic interaction map reveals pathways underlying ricin susceptibility. Cell. 2013; 152(4): 909–22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSims D, Mendes-Pereira AM, Frankum J, et al.: High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing. Genome Biol. 2011; 12(10): R104. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim J, Tan AC: BiNGS!SL-seq: a bioinformatics pipeline for the analysis and interpretation of deep sequencing genome-wide synthetic lethal screen. Methods Mol Biol. 2012; 802: 389–98. PubMed Abstract \| Publisher Full Text\n\nRobinson MD, McCarthy DJ, Smyth GK: edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010; 26(1): 139–40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGentleman RC, Carey VJ, Bates DM, et al.: Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 2004; 5(10): R80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nR Core Team. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria, 2014. Reference Source\n\nRobinson MD, Smyth GK: Small-sample estimation of negative binomial dispersion, with applications to sage data. Biostatistics. 2008; 9(2): 321–32. PubMed Abstract \| Publisher Full Text\n\nMcCarthy DJ, Chen Y, Smyth GK: Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Res. 2012; 40(10): 4288–97. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu D, Lim E, Vaillant F, et al.: ROAST: rotation gene set tests for complex microarray experiments. Bioinformatics. 2010; 26(17): 2176–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu D, Smyth GK: Camera: a competitive gene set test accounting for inter-gene correlation. Nucleic Acids Res. 2012; 40(17): e133. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRitchie ME: Analysing shRNA-seq data using edgeR, supplementary data and documentation. 2014. Reference Source\n\nGiardine B, Riemer C, Hardison RC, et al.: Galaxy: a platform for interactive large-scale genome analysis. Genome Res. 2005; 15(10): 1451–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGoecks J, Nekrutenko A, Taylor J: The Galaxy Team. Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol. 2010; 11(8): R86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlankenberg D, Von Kuster G, Nathaniel C, et al.: Galaxy: a web-based genome analysis tool for experimentalists. Curr Protoc Mol Biol. 2010; Chapter 19: 19.10.1–21. PubMed Abstract \| Publisher Full Text\n\nWang T, Wei JJ, Sabatini DM, et al.: Genetic screens in human cells using the CRISPR/Cas9 system. Science. 2014; 343(6166): 80–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShalem O, Sanjana NE, Hartenian E, et al.: Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. 2014; 343(6166): 84–7. PubMed Abstract \| Publisher Full Text" }	[ { "id": "4546", "date": "01 May 2014", "name": "James W. MacDonald", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a useful manuscript, detailing the author's extensions of existing functionality within the Bioconductor edgeR package to include shRNA screen data.The manuscript itself provides an overview of shRNA screens, competing analysis pipelines, and the methods available in edgeR. In addition, the authors provide a link to a vignette that gives example analyses of four different shRNA experiments that vary in depth of sequencing and complexity, along with the data so potential users can recapitulate the analyses provided, before making an attempt to analyze their own data.", "responses": [] }, { "id": "4552", "date": "08 May 2014", "name": "Ross Lazarus", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well written paper describing a potentially useful application for the analysis shRNA-seq screening data. It includes a brief description of this relatively novel technique and some competing methods as well as a description of the method used in this implementation. The authors are to be commended for providing a wrapper for Galaxy which will make it very easy for biologists to access the method in a reproducible analysis environment and this seems likely to improve the real availability and eventual impact of their work.", "responses": [] }, { "id": "4548", "date": "12 May 2014", "name": "Sumit Deswal", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors introduce the multiplex shRNA screening approach as commonly used currently to find new drug targets or other similar applications. They also provide a literature overview on the relevant bioinformatics tools used in this context. With rapid improvements in the shRNA technology, data analysis methods for this are high in demand. The manuscript provides a good framework for analysis of data on pooled shRNA screens. There is a strong need to have such user friendly, open-source programs dedicated to analysis of shRNA screens that can easily be used by experimental biologists. The manuscript provides a coherent platform that nicely incorporates analytic tools for the current requirements of the RNAi or other similar functional genetic screens. As part of the review process, we have analyzed our own data using the software and found it fully functional and very easy to use. Based on our experience, we recommend the manuscript for indexation.", "responses": [] } ]	1	https://f1000research.com/articles/3-95
https://f1000research.com/articles/3-247/v1	20 Oct 14	{ "type": "Observation Article", "title": "Differential recolonization of Atlantic intertidal habitats after disturbance reveals potential bottom-up community regulation", "authors": [ "Willy Petzold", "Ricardo A. Scrosati", "Willy Petzold" ], "abstract": "In the spring of 2014, abundant sea ice that drifted out of the Gulf of St. Lawrence caused extensive disturbance in rocky intertidal habitats on the northern Atlantic coast of mainland Nova Scotia, Canada. To monitor recovery of intertidal communities, we surveyed two wave-exposed locations in the early summer of 2014. Barnacle recruitment and the abundance of predatory dogwhelks were low at one location (Tor Bay Provincial Park) but more than 20 times higher at the other location (Whitehead). Satellite data indicated that the abundance of coastal phytoplankton (the main food source for barnacle larvae) was consistently higher at Whitehead just before the barnacle recruitment season, when barnacle larvae were in the water column. These observations suggest bottom-up forcing of intertidal communities. The underlying mechanisms and their intensity along the NW Atlantic coast could be investigated through studies done at local and regional scales.", "keywords": [ "The NW Atlantic coast displays cold-temperate intertidal environments. In Nova Scotia (Canada) in winter", "ice does not form on the sea surface on the open Atlantic coast. However", "sea ice readily forms in relatively enclosed water bodies such as gulfs1", "causing physical disturbance on intertidal communities as the ice moves with tides", "currents", "waves", "and wind2", "3. In particular", "abundant sea ice forms every winter on the large Gulf of St. Lawrence (Canadian Ice Service). Between late winter and early spring", "fragments of sea ice drift out of the gulf through the Cabot Strait (between Nova Scotia and Newfoundland) towards the open ocean. Such drift ice then travels south following the open Atlantic coast of Nova Scotia (Figure 1)", "reaching different distances every year depending on the ice load (Canadian Ice Service)." ], "content": "Observation\n\nThe NW Atlantic coast displays cold-temperate intertidal environments. In Nova Scotia (Canada) in winter, ice does not form on the sea surface on the open Atlantic coast. However, sea ice readily forms in relatively enclosed water bodies such as gulfs1, causing physical disturbance on intertidal communities as the ice moves with tides, currents, waves, and wind2,3. In particular, abundant sea ice forms every winter on the large Gulf of St. Lawrence (Canadian Ice Service). Between late winter and early spring, fragments of sea ice drift out of the gulf through the Cabot Strait (between Nova Scotia and Newfoundland) towards the open ocean. Such drift ice then travels south following the open Atlantic coast of Nova Scotia (Figure 1), reaching different distances every year depending on the ice load (Canadian Ice Service).\n\nThe two studied locations on the Atlantic coast of mainland Nova Scotia are indicated with black dots. The arrows indicate the direction that the sea ice from the Gulf of St. Lawrence normally follows when drifting out of the gulf. The asterisk shows the southernmost reach of the drift ice on the Atlantic coast during the 2014 ice season, according to the Canadian Ice Service.\n\nThe open Atlantic coast of mainland Nova Scotia (Figure 1) is reached by drift ice only in some years, more often in northern sections of this coast because of their closer proximity to the Cabot Strait (Canadian Ice Service). In the early spring of 2014, large amounts of sea ice drifted out of the Gulf of St. Lawrence and, during the first half of April, reached up to 92 km of the northern open coast of mainland Nova Scotia. Just before the arrival of the ice, seaweeds and invertebrates were abundant in many rocky intertidal communities4, as drift ice had not reached that coast for the previous 3–4 years (Canadian Ice Service). However, after the ice scoured intertidal habitats for days (up to 16 days at the northern end of this coastal range), intertidal biomass losses were high. For example, in wave-exposed habitats where algal canopies and sessile invertebrates (barnacles and mussels) were abundant before the arrival of the ice, only bare rock was visible after ice scour4.\n\nTo evaluate recolonization patterns, in the summer of 2014 we surveyed two wave-exposed locations that had been heavily scoured by ice in early April4: Whitehead (45.212° N, 61.174° W) and Tor Bay Provincial Park (45.183° N, 61.355° W; Figure 1). The surveyed intertidal habitats face the open Atlantic Ocean directly. On 23 June 2014, at each location we measured the density of barnacle recruits (Semibalanus balanoides) in 8 quadrats (10 cm × 10 cm) that we had randomly established along 30-m transect lines at the mid-to-high intertidal zone in late April. Because of the intense ice scour in early April, macroscopic organisms were absent at this zone in late April, so the substrate was then fully available for barnacle recruitment (barnacles are often the first sessile invertebrates to recolonize disturbed intertidal habitats5,6).\n\nSemibalanus balanoides is the only species of intertidal barnacle on this coast1. Every year, recruits of S. balanoides accumulate in intertidal habitats on this coast during May and June7,8. Our measurements (Dataset 1) on 23 June (after which no new recruits appeared) indicated that barnacle recruit density was significantly higher (Student’s t14 = 3.10, P = 0.017) at Whitehead (199.8 ± 62.0 recruits dm-2, mean ± SE, n = 8 quadrats; Figure 2) than at Tor Bay Provincial Park (7.3 ± 4.2 recruits dm-2; Figure 3). This statistical test was performed in Excel 2004 for Mac. No other sessile macroscopic species occurred at that time in the quadrats.\n\nPicture taken at low tide on 23 June 2014 at the mid-to-high intertidal zone at a wave-exposed habitat in Whitehead, showing many barnacle recruits on the substrate. The inner boundary of the depicted PVC quadrat is 10 cm × 10 cm.\n\nPicture taken at low tide on 23 June 2014 at the mid-to-high intertidal zone at a wave-exposed habitat in Tor Bay Provincial Park, showing very few barnacle recruits on the substrate. The inner boundary of the depicted PVC quadrat is 10 cm × 10 cm.\n\nThe greater density of barnacle recruits at Whitehead than at Tor Bay Provincial Park was related to a higher nearshore chlorophyll-a concentration during late March and April at Whitehead, according to MODIS satellite data (Table 1; National Aeronautics and Space Administration). Nearshore chlorophyll-a concentration indicates coastal phytoplankton abundance, and phytoplankton is the main food source for barnacle nauplius larvae9. For S. balanoides from the Atlantic coast of Nova Scotia, nauplius larvae occur in coastal waters for 5–6 weeks before metamorphosis to cyprids and then intertidal settlement10, which starts in early May on our studied coast8. Thus, it is possible that the higher food supply for larvae at Whitehead than at Tor Bay Provincial Park may have ultimately contributed to determining the higher barnacle recruitment at Whitehead. A positive relationship between nearshore phytoplankton abundance and intertidal barnacle recruitment was previously documented for NW Atlantic intertidal systems at a regional scale7.\n\nTo see whether barnacle recruitment could influence higher trophic levels, we measured the abundance of dogwhelks (Nucella lapillus; Figure 4) shortly after the end of the barnacle recruitment season. Nucella lapillus is the main predator of barnacles on the studied coast1, so presumably a higher barnacle recruitment could locally increase dogwhelk abundance. On 15 July 2014, at each of the two studied locations we measured during low tide the density of N. lapillus in 30 quadrats (50 cm × 50 cm) randomly established at the mid-to-high intertidal zone (Dataset 2). Dogwhelk density was significantly higher (Student’s t58 = 2.64, P = 0.013) at Whitehead (39.6 ± 14.2 individuals m-2, mean ± SE, n = 30 quadrats) than at Tor Bay Provincial Park (1.9 ± 0.8 individuals m-2). Barnacle recruitment in June 2014 may not fully explain dogwhelk density in July 2014, as dogwhelks had not undergone their 2014 recruitment season as yet (mainly in late summer11). However, visits to both studied locations in 2012 and 2013 revealed a similar difference in barnacle recruitment between both locations (R.A.S., pers. obs.), supporting the notion that dogwhelk abundance may be driven by barnacles on this coast. Interestingly, in 2014, barnacle recruits and dogwhelks were more abundant at Whitehead than at Tor Bay Provincial Park by a similar ratio (27.6 times higher for barnacles and 20.8 times higher for dogwhelks), further suggesting a possible dependency of dogwhelk abundance on barnacle recruitment.\n\nPicture taken at low tide on 15 July 2014 at the mid-to-high intertidal zone at a wave-exposed habitat in Whitehead, showing dogwhelks foraging on the bed of barnacle recruits. A few barnacle shells appear empty likely as a result of recent dogwhelk predation.\n\nRelationships between coastal chlorophyll-a concentration, intertidal barnacle recruitment, and intertidal predator impacts have been identified on Pacific rocky shores6. The positive influence of prey food supply on predators mediated by prey recruitment is referred to as bottom-up regulation of community structure12. Coastal configuration and water column movements influence nearshore phytoplankton abundance6. What caused the phytoplankton difference between our two studied locations remains to be determined. However, the observed link between phytoplankton abundance, barnacle recruitment, and dogwhelk density does suggest that bottom-up forcing may also structure NW Atlantic intertidal communities. Understanding the underlying mechanisms and their intensity along the coast could be achieved with a larger spatial monitoring and field experimentation.\n\n\nData availability\n\nF1000Research: Dataset 1. Abundance of barnacle recruits at the end of the 2014 recruitment season, 10.5256/f1000research.5545.d3709313\n\nF1000Research: Dataset 2. Abundance of dogwhelks shortly after the 2014 barnacle recruitment season, 10.5256/f1000research.5545.d3709414", "appendix": "Author contributions\n\n\n\nWP and RAS did the field surveys. RAS wrote the manuscript and WP provided critical comments to produce the final version.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was funded by a Discovery Grant (# 311624) awarded to R.A.S. by the Natural Sciences and Engineering Research Council of Canada (NSERC).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nScrosati R, Heaven C: Spatial trends in community richness, diversity, and evenness across rocky intertidal environmental stress gradients in eastern Canada. Mar Ecol Prog Ser. 2007; 342: 1–14. Publisher Full Text\n\nScrosati R, Heaven C: Field technique to quantify intensity of scouring by sea ice in rocky intertidal habitats. Mar Ecol Prog Ser. 2006; 320: 293–295. Publisher Full Text\n\nBelt KM, Cole SW, Scrosati RA: Intertidal barnacles as indicators of the intensity of scour by sea ice. Mar Ecol Prog Ser. 2009; 381: 183–187. Publisher Full Text\n\nPetzold W, Willers MT, Scrosati RA: Visual record of intertidal disturbance caused by drift ice in the spring on the Atlantic coast of Nova Scotia. F1000Res. 2014; 3: 112. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMinchinton TE, Scheibling RE, Hunt HL: Recovery of an intertidal assemblage following a rare occurrence of scouring by sea ice in Nova Scotia, Canada. Bot Mar. 1997; 40: 139–148. Publisher Full Text\n\nMenge BA, Menge DNL: Dynamics of coastal meta-ecosystems: the intermittent upwelling hypothesis and a test in rocky intertidal regions. Ecol Monogr. 2013; 83: 283–310. Publisher Full Text\n\nCole SWB, Scrosati RA, Tam JC, Sussmann AV: Regional decoupling between NW Atlantic barnacle recruit and adult density is related to changes in pelagic food supply and benthic disturbance. J Sea Res. 2011; 65(1): 33–37. Publisher Full Text\n\nBeermann AJ, Ellrich JA, Molis M, Scrosati RA: Effects of seaweed canopies and adult barnacles on barnacle recruitment: the interplay of positive and negative influences. J Exp Mar Biol Ecol. 2013; 448: 162–170. Publisher Full Text\n\nAnderson DT: Barnacles. Structure, Function, Development, and Evolution. Chapman & Hall, London, UK. 1994. Reference Source\n\nBousfield EL: The distribution and spawning seasons of barnacles on the Atlantic coast of Canada. Bull Natl Mus Canada. 1954; 132: 112–154.\n\nHunt HL, Scheibling RE: Effects of whelk (Nucella lapillus (L.)) predation on mussel (Mytilus trossulus (Gould), M. edulis (L.)) assemblages in tidepools and on emergent rock on a wave-exposed rocky shore in Nova Scotia, Canada. J Exp Mar Biol Ecol. 1998; 226(1): 87–113. Publisher Full Text\n\nMoore JC, de Ruiter PC: Bottom-up control. In: Hastings A, Gross LJ, editors; Encyclopedia of Theoretical Ecology, University of California Press, Berkeley, USA. 2012. Reference Source\n\nPetzold W, Scrosati RA: Dataset 1. Abundance of barnacle recruits at the end of the 2014 recruitment season. F1000Research. 2014. Data Source\n\nPetzold W, Scrosati RA: Dataset 2. Abundance of dogwhelks shortly after the 2014 barnacle recruitment season. F1000Research. 2014. Data Source" }	[ { "id": "6458", "date": "23 Oct 2014", "name": "Javier Calcagno", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study is an interesting first approach to understanding the factors that affect the ability to recruit sessile benthic organisms after disturbances in an intertidal community. Petzold and Scrosati address the issue concerning the role of larval supply and its relation to phytoplankton abundance, which leads directly to considering that a bottom up mechanism may be acting in these communities. Furthermore, the idea that began the work is clearly stated and the theoretical context is appropriate, as well as the statistical treatment of the data. Although a more deep and permanent approach to the problem requires the carrying out of further studies, perhaps with the incorporation of field experiments, this initial statement is correct and I recommend its indexing.", "responses": [] }, { "id": "9458", "date": "13 Jul 2015", "name": "Nelson Valdivia", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, Petzold and Scrosati provide observational evidence suggesting that food supply drives the re-colonisation of benthic organisms in wave-exposed intertidal communities after being disturbed by drifting ices. The title and abstract of the article are appropriate for the content of the work and represent a suitable summary of it. Although the authors report the results of only one site for each “condition” (i.e. one site with high and one site with low Chlorophyll-a concentration), the differences in food supply between these sites are strong enough to propose further studies analysing the role of bottom-up forcing in this system. Accordingly, the conclusions of the study are sensible and justified on the basis of the results. As a follow-up approach, the authors may consider first, to expand the study to a larger set of sites in order to confirm the pattern, and second, to construct competing hypotheses (in addition to the “bottom-up hypothesis”) that would be tested by means of manipulative experiments replicated at those sites. This article is a necessary benchmark from which further hypothesis-driven research should be conducted", "responses": [] } ]	1	https://f1000research.com/articles/3-247
https://f1000research.com/articles/3-180/v1	31 Jul 14	{ "type": "Observation Article", "title": "Thrombocytosis portends adverse prognostic significance in patients with stage II colorectal carcinoma", "authors": [ "Tianhua Guo", "Marcin Krzystanek", "Zoltan Szallasi", "Arpad Szallasi", "Tianhua Guo", "Marcin Krzystanek", "Zoltan Szallasi" ], "abstract": "Thrombocytosis portends adverse prognostic significance in many types of cancers including ovarian and lung carcinoma. In this study, we determined the prevalence and prognostic significance of thrombocytosis (defined as platelet count in excess of 400 K/μl) in patients with colorectal cancer. We performed a retrospective analysis of 310 consecutive patients diagnosed at our institution between 2004 and 2013. The patients (48.7% male and 51.3% female) had a mean age of 69.9 years (+/- 12.7 years) at diagnosis. Thrombocytosis was found in a total of 25 patients, with a higher incidence in those with stage III and IV disease (14.4% of patients). Although the mean platelet count increased with the depth of tumor invasion (pT), its values remained within normal limits in the whole patient cohort. No patient with stage I cancer (n=57) had elevated platelet count at diagnosis. By contrast, five of the 78 patients (6.4%) with stage II cancer showed thrombocytosis, and four of these patients showed early recurrence and/or metastatic disease, resulting in shortened survival (they died within one year after surgery). The incidence of thrombocytosis increased to 12.2% and 20.6%, respectively, in patients with stage III and IV disease. The overall survival rate of patients with thrombocytosis was lower than those without thrombocytosis in the stage II and III disease groups, but this difference disappeared in patients with stage IV cancer who did poorly regardless of their platelet count. We concluded that thrombocytosis at diagnosis indicates adverse clinical outcome in colorectal cancer patients with stage II or III disease. This observation is especially intriguing in stage II patients because the clinical management of these patients is controversial. If our data are confirmed in larger studies, stage II colon cancer patients with thrombocytosis should be upstaged and treated as stage III/IV disease patients.", "keywords": [ "Platelets play important roles in hemostasis", "immunity and inflammation1. Cancer is often associated with thrombocytosis2. Thrombocytosis was reported as a poor prognostic indicator in many types of cancers including lung cancer3", "renal cell carcinoma4 and gynecological cancers5. A positive correlation between the depth of tumor invasion and platelet counts was demonstrated in a gastric cancer study", "and thrombocytosis served as an adverse prognostic factor in clinical outcome in gastric cancer patients6. Recent studies have shown that thrombocytosis in cancer may be correlated with serum cytokine levels that stimulate thrombopoiesis. For example", "elevated plasma levels of IL-6 and thrombopoietin were reported in ovarian cancer patients5." ], "content": "Introduction\n\nPlatelets play important roles in hemostasis, immunity and inflammation1. Cancer is often associated with thrombocytosis2. Thrombocytosis was reported as a poor prognostic indicator in many types of cancers including lung cancer3, renal cell carcinoma4 and gynecological cancers5. A positive correlation between the depth of tumor invasion and platelet counts was demonstrated in a gastric cancer study, and thrombocytosis served as an adverse prognostic factor in clinical outcome in gastric cancer patients6. Recent studies have shown that thrombocytosis in cancer may be correlated with serum cytokine levels that stimulate thrombopoiesis. For example, elevated plasma levels of IL-6 and thrombopoietin were reported in ovarian cancer patients5.\n\nColon cancer is a leading cause of cancer-related death in developed countries (http://seer.cancer.gov/statfacts/html/colorect.html). A significant portion of patients receiving potentially curative resection dies within five years of diagnosis7. Since both chemo- and radiation therapies cause very significant side-effects, it is critical to define reliable prognostic factors to identify patients who might benefit from more aggressive adjuvant treatment options. The prognostic role of thrombocytosis in colorectal cancer patients has not been fully investigated, although there are some reports supporting the negative impact of thrombocytosis on the survival of patients with colorectal cancer8,9.\n\nIn this study, the aim was to analyze the association between platelet count at diagnosis or pre-surgery and cancer stage, and to determine the prognostic significance of thrombocytosis in patients with colorectal cancer.\n\n\nPatients and methods\n\nAfter approval by the Monmouth Medical Center Institutional Research Review Board (IRB Study # 213-041), the medical records of 310 consecutive colorectal cancer patients who underwent biopsy (47 patients), surgical resection and/or neoadjuvant treatment (263 patients) at our institution between 2004 and 2013 were retrospectively reviewed. The patient cohort included 62 patients with in-situ or stage I disease, 78 patients at stage II, 98 patients at stage III, 34 patients at stage IV, and 38 patients with biopsy diagnosis only (stage could not be determined). The mean age of the patients was 69.9±12.7 years (range = 32 to 98 years). The data gathered included platelet counts, tumor location, histological type, lymph node metastasis, depth of tumor invasion (T), presence or absence of distant metastasis, and survival time.\n\nPre-operative platelet counts were collected from our Laboratory Information System (LIS), and thrombocytosis was defined as platelet count ≥ 400 × 103/μl. After resection of the tumor, all specimens were histologically examined by a pathologist and the pathological TNM stage was determined according to the American Joint Committee on Cancer, 7th edition. Stage I and II cancers are lymph node negative (N0) whereas stage III is defined by the presence of lymph node metastasis. Patients with distant metastatic disease are classified as stage IV.\n\nSurvival data were provided by the Cancer Registry at the Leon Hess Cancer Center, Monmouth Medical Center. All calculations were made using R version 2.15.0 and packages “beeswarm”, “survplot”, “survival” and “stats”. Survival curves were generated using the Kaplan-Meier method. Hazard ratios with 95% confidence intervals were obtained using Cox proportional hazards regression. Long-rank test was used for the analysis of significance.\n\n\nResults\n\nThe characteristics (including age, gender, pT stage, tumor differentiation and platelet count) of the patients in our study cohort are shown in Table 1. Of the 310 patients with colorectal cancer, 25 (8.1%) had thrombocytosis at diagnosis or pre-surgery with the highest incidence detected in stage IV patients (20.6%) [Table 2]. Importantly, none of the 57 patients with stage I carcinoma had elevated platelet count. Although the mean platelet count increased with the depth of invasion (pT), it remained within the normal limits in all patients groups (pT1 to pT4) [Figure 1]. Mean platelet counts (×103/µL) were 216±71 (pT1), 252±83 (pT2), 274±109 (pT3), and 291±104 (pT4); this increase was significant at P = 0.001. By contrast, there were no significant differences in thrombocytosis with regard to gender, age, location, or tumor differentiation (data not shown).\n\na: Data presented as mean ± SD; b: staging according to AJCC Cancer Staging Manual 7th Edition); c: Grading according to WHO grading system\n\nNote: There are 403 cases in our data pool. 310 cases were diagnosed as colon cancer among the 321 cases with available platelet counts (82 cases without platelet counts). 11 non-colorectal cancer cases including 5 cases labeled as “appendix” and 6 cases labeled as “not colon cancer” were taken out of the pool.\n\nThe mean platelet count of patients with pT1, (216±71) × 103/µL is significantly lower than patients with pT4 (291±104) × 103/µL, P = 0.001.\n\nThe combined incidence of thrombocytosis in stage III and IV disease was 14.4%. Stage II disease had the lowest incidence (6.4%) and stage IV cancer showed the highest incidence of elevated platelet count (20.6%) [Table 2]. The overall survival of patients with stage I to stage III colorectal cancer with thrombocytosis was significantly lower than those without thrombocytosis [Figure 2]. Although patients with Stage IV carcinoma had the highest prevalence of thrombocytosis, these patients did uniformly poorly and the difference in survival was no longer observed in patients with or without elevated platelet count (not shown). Patients with thrombocytosis (PLT ≥ 400 × 103/μL) at stage I to stage III had a hazard ratio of 2.2 compared to the patients without thrombocytosis (PLT < 400 × 103/μL) as shown in Figure 2.\n\nPatients were allocated into two groups: with thrombocytosis, PLT ≥ 400 × 103/µL (labeled as Exceeds) and without thrombocytosis) PLT < 400 × 103/µL (labeled as Normal). The patients with thrombocytosis have a hazard ratio of 2.2 compare to the patients without thrombocytosis (P<0.05).\n\n\nDiscussion\n\nIt was noted more than 100 years ago that thrombocytosis is often seen in patients with malignant diseases. Indeed, thrombocytosis correlates with both worse disease free survival and shortened overall survival in patients with ovarian cancer3, and worsens overall survival in patients with gastric cancer (reviewed in 10). The prognostic significance of thrombocytosis in colorectal cancer, however, remains controversial11, although the majority of literature suggests a negative impact of thrombocytosis on the survival of patients with colorectal cancer8,9.\n\nIn the present study, we identified a mild, T stage-dependant increase in mean platelet counts in patients with colorectal carcinoma that reached statistical significance when comparing T1 to T4; however, the mean platelet count remained within the normal limits in the whole patient cohort. Importantly, thrombocytosis was more common among patients with advanced disease: its prevalence increased from 6.4% in stage II to 20.6% in stage IV cancer patients. Our data are comparable to those reported in the literature (12.1–13.9%;6,7).\n\nIn our study, thrombocytosis showed adverse prognostic significance in patients with stage I to stage III colorectal carcinoma; this was no longer apparent in patients with stage IV disease, presumably because these patients did poorly regardless of the platelet count.\n\nProbably the most intriguing observation of our study is the fairly uniformly dismal clinical outcome of stage II patients with thrombocytosis (five out of 78 patients): four of these five patients did very poorly (they died within a year) and the fifth was lost to follow-up. We suspect that these cases might represent stage III/IV cases misclassified as stage II due to false negative lymph node examination. At present, the National Comprehensive Cancer Network guidelines do not recommend routine administration of adjuvant chemotherapy to stage II colorectal cancer patients whose cancer was completely resected12. If our data are confirmed in future larger studies, stage II colorectal cancer patients with thrombocytosis should be upstaged and treated clinically as stage III/IV.\n\nThe molecular mechanisms underlying thrombocytosis in cancer patients are incompletely understood. Recently, plasma levels of IL-6 and thrombopoietin were found to significantly correlate with platelet counts in ovarian cancer patients. Indeed, silencing of the IL-6 and thrombopoietin genes markedly abrogated thrombocytosis (and halted tumor progression) in a mouse model of epithelial ovarian cancer5. These findings are promising because an anti-IL-6 agent (siltuximab) has already been approved by the United States Food and Drug Administration (FDA) to treat patients with Castleman’s disease (http://www.cancer.org//cancer/new/fda-approves-sylvant-siltuximab-for-castleman-disease). Another series of study showed that immobilized platelets support human colon carcinoma cell tethering, rolling, and firm adhesion under dynamic flow conditions in colon cancer cell lines, suggesting a role of platelets in hematogenous dissemination of tumor cells in colorectal cancer13. Taken together, these studies imply that inhibition of thrombocytosis may represent a potential novel target in cancer therapy.\n\nIn summary, thrombocytosis appears to herald adverse clinical outcome in patients with stage II and III colorectal carcinoma. We suggest that elevated platelet count may identify a subset of patients with stage II colon cancer who could benefit from close follow-up and aggressive adjuvant therapy.\n\n\nData availability\n\nF1000Research: Dataset 1. Data of thrombocytosis in colon cancer patients, 10.5256/f1000research.4856.d3337114", "appendix": "Author contributions\n\n\n\nTianhua Guo: collected data.\n\nMarcin Krzystanek: analysed data.\n\nZoltan Szallasi: initiated project, supervised data analysis.\n\nArpad Szallasi: supervised project, prepared MS.\n\nAll authors revised the manuscript and agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this study.\n\n\nReferences\n\nMichelson AD (ed): Platelets. Academic Press, Ebook. 2011. Reference Source\n\nLin RJ, Afshar-Khargan V, Schafer AI: Paraneoplastic thrombocytosis: the secrets of tumor self-promotion. Blood. 2014; 124(2): 184–187. PubMed Abstract \| Publisher Full Text\n\nCostatini V, Zacharski LR, Moritz TE, et al.: The platelet count in carcinoma of the lung and colon. Thromb Haemost. 1990; 64(4): 501–505. PubMed Abstract\n\nSymbas NP, Townsend MF, El-Galley R, et al.: Poor prognosis associated with thrombocytosis in patients with renal cell carcinoma. BJU Int. 2000; 86(3): 203–207. PubMed Abstract \| Publisher Full Text\n\nStone RL, Nick AM, McNeish IA, et al.: Paraneoplastic thrombocytosis in ovarian cancer. N Eng J Med. 2012; 366(7): 610–618. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIkeda M, Furukawa H, Imamura H, et al.: Poor prognosis associated with thrombocytosis in patients with gastric cancer. Ann Surg Oncol. 2002; 9(3): 287–291. PubMed Abstract\n\nMcArdle CS, Hole DJ: Outcome following surgery for colorectal cancer. Br Med Bull. 2002; 64: 119–125. PubMed Abstract \| Publisher Full Text\n\nSasaki K, Kawai K, Tsuno NH, et al.: Impact of preoperative thrombocytosis on the survival of patients with primary colorectal cancer. World J Surg. 2012; 36(1): 192–200. PubMed Abstract \| Publisher Full Text\n\nKandemir EG, Mayadagli A, Karagoz B, et al.: Prognostic significance of thrombocytosis in node-negative colon cancer. J Int Med Res. 2005; 33(2): 228–235. PubMed Abstract \| Publisher Full Text\n\nVoutsadakis IA: Thrombocytosis as a prognostic marker in gastrointestinal cancers. World J Gastrointest Oncol. 2014; 6(2): 34–40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNyasavajjala SM, Runau F, Datta S, et al.: Is there a role for pre-operative thrombocytosis in the management of colorectal cancer? Int J Surg. 2010; 8(6): 436–438. PubMed Abstract \| Publisher Full Text\n\nEngstrom PF, Arnolett JP, Benson AB 3rd, et al.: NCCN Clinical Practice Guidelines for Oncology: colon cancer. J Natl Compr Canc Netw. 2009; 7(8): 778–831. PubMed Abstract\n\nMcCarty OJ, Mousa SA, Bray PF, et al.: Immobilized platelets support human colon carcinoma cell tethering, rolling, and firm adhesion under dynamic flow conditions. Blood. 2000; 96(5): 1789–1797. PubMed Abstract\n\nGuo T, Krzystanek M, Szallasi Z, et al.: Data of thrombocytosis in colon cancer patients. F1000Research. 2014. Data Source" }	[ { "id": "5655", "date": "14 Aug 2014", "name": "Ioannis A. Voutsadakis", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting retrospective study of an extensive series of colorectal cancer patients and authors are to be congratulated on undertaking this academic endeavour. Nevertheless there are several points that need to be addressed. A major concern is that all stages of the disease have been included. In general a comparison between stage I and IV, for example, has no meaning clinically and the only statement that one can make is that thrombocytosis is more common. Another major concern is that no multivariate analysis is done or presented.Some other specific points:The title mentions specifically stage II disease (as the authors probably recognize the importance of prognostic factors in this sub-group for therapeutic decisions) but no statistical comparison is offered anywhere in the article. In any case the number of patients with thrombocytosis in this group (5) is prohibitive for definite conclusions. In the last line of the Abstract and in the Discussion it should be: “treated as stage III” or even better “considered for adjuvant chemotherapy” (stage IV patients are treated with palliative intent). Data on tumor grade and location have been collected (dataset) but not summarized or presented in the article. It would be of particular interest to present and include in an eventual multivariate analysis tumor location (right versus left from the splenic flexure versus rectal). In Patients and methods the normal range of platelets in authors’ institution should be mentioned. In the Results it would be interesting to provide the mean and SD of the platelet number in the 2 groups (normal and thrombocytosis). In fig. 2 there is a discrepancy between HR and p values in the body and the legend. In addition the numbers at risk do not match. Is that a mistake in calculations or data missing? Authors should address this. In the 4th line of the 1st paragraph of the Discussion” the reference does not seem to be correct. Same with reference 6 in the 2nd paragraph. In the discussion on metastatic patients - this is an over-simplification. If any useful comparison is to be made, other data have to be entered in the equation such as oligometastatic versus polymetastatic disease and treatments. Treatments should be mentioned. For example have all stage III patients received adjuvant chemotherapy? And have any of the stage II patients received any such treatment? In the 4th paragraph of the Discussion the explanation of “false negative node examination” is improbable as there is no reason to suspect that such false negative occurs more commonly in patients with thrombocytosis. Instead a difference in biology as briefly discussed in the next paragraph is more probable.", "responses": [ { "c_id": "1027", "date": "13 Oct 2014", "name": "Arpad Szallasi", "role": "Author Response", "response": "We are grateful for the referee for his thoughts in making this a better MS. Whereas we agree with most of the reservations of the referee, we would like to point out that this is an Observation Study, and not a full report. We believe that despite the limitations of our study, our conclusions are sound and (if confirmed) may have important implications for patient management.Major changes done in response to points raised by the referee include:We agree with the referee that the small number of patients (5) in the stage II group precludes a definitive conclusion; this is now clearly stated in the MS. Again, this is an Observational Study within the confines of our hospital database on colorectal carcinoma patients. Both the Abstract and the Discussion have been modified as suggested by the referee (\"treated as stage III\" has been changed to \"may be considered for adjuvant chemotherapy\"). The multivariate analysis with special regard to the location of the tumor (right-sided versus left-sided) in now detailed in the Results. The normal range of platelets at our Institution (150 to 400 K/ul) is now provided in the MS. The mean +/-SD of the platelet counts in the two patient groups (with and without thrombocytosis) is now provided in Results. Indeed, there is a discrepancy between the body of the text and Figure 2 with regard to the patient numbers; this should have been addressed before to avoid any confusion. As clearly stated now in Patients and Methods, our database included 310 patients. Of these patients, our Cancer Registry had long-term survival data on 253 patients (hence the discrepancy): the remaining 57 patients most likely decided to seek treatment at a different facility. References have been corrected. Discussion on metastatic patients has been modified. Unfortunately, our Cancer Registry has no full details on chemotherapy regimens (some of our patients receive chemotherapy in a private practice setting). The reference to \"false negative node examination\" has been eliminated." } ] }, { "id": "6229", "date": "06 Oct 2014", "name": "Judit Moldvay", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is an interesting work providing new information on the issue of thrombocytosis in colon cancer patients. It contains relevant clinicopathologic parameters, however, there is no data on ethnicity, smoking habits, inflammatory processes, comorbidities, or medications of the studied patients, all which might have impact on platelet count (Msaouel et al., 2014).The main factors that can influence the platelet count should at least be discussed.In Figure 1. the Authors discussed the mean platelet count according to the depth of tumor invasion. Although it might change statistically significantly, the mean platelet count remains much within the normal range, therefore, the conclusion that “… we identified a mild, T stage-dependant increase in mean platelet count …” is not well-founded. One of the most critical aspects of the investigation is the number of patients, especially in stage II, as disproportionately firm conclusions have been drawn regarding correlation between overall survival and thrombocytosis on the basis of only four patients with thrombocytosis in stage II.Another weakness of the study is the lack of information on the precise localizations of the primary tumors, as this in itself may be of prognostic value, e.g. Majek et al. (2012) .Although neoadjuvant treatment has been mentioned, it was not discussed in details, similarly, adjuvant treatments in advanced cases were not detailed either.These issues should be discussed in the \"Discussion\".In Figure 2. the value of HR is not in accordance with the HR in the legend.In the 4th line of the Discussion there is a mistake with the Ref. 3., this – most probably – is the Ref. 5.Similarly, in the 18th line of the Discussion the Ref. 6. – most probably – is the Ref. 8.In conclusion, this study yielded clinically very interesting results that might have therapeutic consequence, however, these results need to be confirmed in a much larger cohort of colorectal patients.All in all, in my opinion this manuscript is acceptable for indexation, but only after corrections and completions based on the above mentioned comments.", "responses": [ { "c_id": "1026", "date": "13 Oct 2014", "name": "Arpad Szallasi", "role": "Author Response", "response": "Whereas we share some of the reservations of the referee, we would like to point out that this is an Observational Study and not a full report. We believe that despite the limitations of this study, this is an interesting observation with potential implications for patient management. That said, we wholeheartedly agree with the referee that \"these results need to be confirmed in a much larger cohort of colorectal patients.\" Changes done in response to points raised by the referee in the revised MS include:Our study has a number of limitations such the lack of data on ethnicity, smoking habits, comorbidities, or medications: this is now clearly stated in Patients and Methods. We believe that such a detailed, multifactorial analysis would surpass the confines of a preliminary Observational Report. Of note, the patient database is available for review for the interested reader (please follow link under Reference 14 to \"data source\"). The most common causes of reactive thrombocytosis are now briefly listed in the Introduction. The statement between platelet count and depth of invasion has been toned down - the reference to significance has been eliminated. As emphasized in the MS, our conclusions are preliminary and are based on a limited number of patients. It is our hope that our findings will be confirmed (or refuted) based on a much larger patient cohort. The localization of the primary tumor (right-sided versus left-sided) seems to have no impact on the presence or absence of thrombocytosis: both groups had an incidence of ~7%. This is now clearly stated in Results. References have been corrected." } ] } ]	1	https://f1000research.com/articles/3-180
https://f1000research.com/articles/3-246/v1	20 Oct 14	{ "type": "Software Tool Article", "title": "Visualisation of BioPAX Networks using BioLayout Express3D", "authors": [ "Derek W. Wright", "Tim Angus", "Anton J. Enright", "Tom C. Freeman", "Tim Angus", "Anton J. Enright", "Tom C. Freeman" ], "abstract": "BioLayout Express3D is a network analysis tool designed for the visualisation and analysis of graphs derived from biological data. It has proved to be powerful in the analysis of gene expression data, biological pathways and in a range of other applications. In version 3.2 of the tool we have introduced the ability to import, merge and display pathways and protein interaction networks available in the BioPAX Level 3 standard exchange format. A graphical interface allows users to search for pathways or interaction data stored in the Pathway Commons database. Queries using either gene/protein or pathway names are made via the cPath2 client and users can also define the source and/or species of information that they wish to examine. Data matching a query are listed and individual records may be viewed in isolation or merged using an ‘Advanced’ query tab. A visualisation scheme has been defined by mapping BioPAX entity types to a range of glyphs. Graphs of these data can be viewed and explored within BioLayout as 2D or 3D graph layouts, where they can be edited and/or exported for visualisation and editing within other tools.", "keywords": [ "signalling network", "analysis", "proteins", "bioinformatics" ], "content": "Introduction\n\nThere has been an explosion in the amount of publicly available pathway and interaction data in recent years, derived from high-throughput experimental techniques, such as two-hybrid systems, mass spectrometry, phage display etc., or through focused studies and manually curated from the literature into pathway models1–3. There are many resources that store such data: at the time of writing, the website pathguide.org4 listed 547 pathway and interaction databases. However, many of these resources store the data in idiosyncratic formats and as a result it has been difficult for resources to exchange data between them.\n\nTo address this problem, there have been a number of efforts to standardize the exchange of pathway and protein interaction data from disparate sources, including PSICQUIC5, CellML6 and BioPAX7. Of these, BioPAX is one of the most widely adopted data exchange formats. BioPAX is a community standard ontology for describing pathway and protein interaction data, suitable for qualitatively representing the current knowledge of biological systems. Seventy-four of the resources listed by the PathGuide currently support BioPAX, including some of the most widely used resources. BioPAX is overseen by the Computational Modeling in Biology Network (COMBINE) (http://co.mbine.org/) and has been released in major versions referred to as levels. The latest release is BioPAX level 3, version 1.0.\n\nPathway Commons8 is a publicly available resource that aggregates and integrates pathway data from multiple organisms and databases into a common BioPAX language linked data representation. Data stored within this resource are currently derived from the databases ChEBI9, UniProt10, Reactome1, Pathway Interaction Database11, PhosphoSite10, HumanCyc12, HPRD13 and PANTHER2. The Pathway Commons website provides query and bulk download of these data. The system also makes these data available via a REST web service API, which provides programmatic access to data over the web. Pathway Commons has recently been upgraded, supporting BioPAX Level 3 and providing advanced graph queries via the CPath2 REST API.\n\nA range of software tools already supports BioPAX use and exchange. For example the network analysis tool Cytoscape14 has support via plugins (known as “apps” in Cytoscape version 3)15,16. The BiNoM plugin17 can import BioPAX Level 3 OWL files, the CyPath2 plugin is able to import and visualise BioPAX from the Pathway Commons resource, and the ChiBE pathway editor18 allows users to visually edit BioPAX pathways. CellDesigner, a graphical pathway editor, can export pathways as BioPAX19 and users of the R statistical programming language can access BioPAX via the rBiopaxParser package20. As BioPAX is a language based on the semantic RDF/OWL standard, it can also be edited using standard ontology authoring tools such as Protégé and WebProtégé21. However, it should be noted that there are various compatibility issues with some of the above, with different apps/tools being specific for different versions of the tools or BioPAX.\n\nThere is considerable interest in BioPAX data from the bioinformatics community and a growing interest in tools that support its visualisation and analysis. Here we report the implementation of a simple-to-use graphical interface within the network analysis tool BioLayout Express3D22,23 that now supports querying of the Pathway Commons resource, allowing the user to pull in the results of specific gene/protein- or pathway-centric queries, and to visualise the results in a graphically intuitive manner.\n\n\nImplementation\n\nBioLayout Express3D version 3.2 has been developed to open BioPAX Level 3 OWL files and generate network graph visualisations of BioPAX encoded pathway or protein interaction data. A web service client has also been developed within BioLayout to query Pathway Commons and import BioPAX networks directly.\n\nPaxTools24, an open-source Java library for developing BioPAX applications, has been incorporated into BioLayout. When a BioPAX OWL file is opened, it is parsed using PaxTools and an in-memory object model containing the elements in the BioPAX document is created. If the BioPAX major version is lower than Level 3, the object model is upgraded to Level 3 before the graph is constructed. The program iterates through each entity, looking up and assigning a shape for that entity type then creating a corresponding graph node. The program then connects nodes by creation of edges to represent components that are members of a complex, steps of a pathway and participants of an interaction.\n\n\nInput options\n\nBioLayout Express3D communicates with Pathway Commons via the cPath2 REST API, sending commands using the PaxTools library. Using a search dialog (Figure 1A), opened by selecting “File -> Network From Public Database…” within BioLayout, the user may refine searches by keywords, species, data source and BioPAX type. For convenience, predefined search options are provided for individual data sources and popular species. Queries may be for specific genes/proteins or for pathways, searching the entire record or just the title.\n\n(A) Import Network search dialog. This dialog supports the querying of the Pathway Commons database. 1. Keywords are entered into the Keywords field. To restrict the search to the BioPAX pathway name only (as opposed to all text associated with pathway), leave the ‘Name’ checkbox ticked. Terms may be separated with Boolean operators AND/OR and a specific search field may be combined with a search term separated by a colon (:) and wildcards may be searched using an asterisk (*); 2. NCBI organism ID number or species name may be entered in the text field or for convenience popular species may be searched for using the checkboxes provided; 3. Information from specific databases from which Pathway Commons aggregates may be selected, restricting searches to information provided by those resources; 4. Dropdown list defines which BioPAX type to search for: Pathway, Interaction, Physical Entity, Entity Reference. There is an additional option, Top Pathways, which is a special case; this is defined as “pathways that are neither ‘controlled’ nor ‘pathwayComponent’ of another process”; 5. Click the Search button to perform the search. Search results are displayed in the table. Results are returned in pages of 500 search hits; the sequence of pages may be navigated using the Next/Previous buttons; 6. Click on a row in the results table to display detailed information about that network in the pane on the right hand side; 7. Click the Open button to download and display the network for the search hit you have highlighted in the results table; 8. If you wish to perform an advanced graph query, double-click the row(s) in the results table and the search hits will be added to the Advanced tab. The Advanced tab of the Import Network dialog enables you to perform advanced graph queries on search merging networks etc.; 9. When the procedure is defined, click Execute to visualise the results.\n\nSearch results are displayed in a table. Clicking on a search hit displays an excerpt from the description with the highlighted search term, the persistent Uniform Resource Identifier (URI) of the search hit on Pathway Commons, the number of interactions (for pathways only) and the species name, which are displayed in a panel alongside the table of hits. As the BioPAX document defines species using Identifiers.org25 standard URIs, the scientific names of the species in the search results are looked up in the NCBI Taxonomy database26 using the NCBI EFetch SOAP27 web service. This live lookup ensures that BioLayout is capable of displaying the name of any species that may be found in the search results. For a pathway search hit, the number of interactions is calculated and displayed, so as to give the user an indication of the size of the network that will be produced (some hits may be very small and possibly not worth displaying). This value is obtained by counting the results of a traverse query of interactions within the selected pathway.\n\nThe user may choose to display the network for a single search hit, in which case BioLayout downloads the corresponding OWL file from Pathway Commons and opens it, displaying the network. Alternatively, a user may choose to select a number of search hits and then perform advanced graph queries on multiple hits, using the operations provided by the Pathway Commons cPath2 web service. A search hit may be added to the Advanced tab by double-clicking the row (Figure 1B). Advanced query options are:\n\nGet – multiple networks combined into a single network\n\nNearest Neighborhood – first order neighborhood of nodes within search hits\n\nCommon Stream – common upstream or downstream of search hits\n\nPaths Between – network forming paths between search hits\n\nPaths From To – network forming the paths from search hits in rows selected by the user in the Advanced table to unselected search hits\n\n\nVisualisation of BioPAX data\n\nA visual scheme has been defined, where node shapes and colours have been mapped to BioPAX entity types. BioLayout already supports the import of mEPN (modified Edinburgh Pathway Notation) pathway models3,28 saved as GraphML files. When visualised, the concepts supported by this pathway notation system are translated into equivalent 2D or 3D shapes. We therefore chose equivalent glyphs for BioPAX entities and concepts where possible, in order to provide a consistent user experience.\n\nSome BioPAX concepts could not be mapped to the existing mEPN scheme so new glyphs were added. For example, a dumbbell shape was added for RNA-Region. Some concepts did not have an exact analogue. In the case of the BioPAX Small Molecule type, the equivalent concept in mEPN could either be Ion/Simple Molecule or Simple Biochemical; the Ion/Simple Molecule glyph was used. The BioPAX ontology has a hierarchical structure with increasing levels of granularity. Some glyphs were added to mEPN to handle generic BioPAX types where the more detailed type is not available in the data, such as a Control transition. Mappings between BioPAX/mEPN concepts and the 2D and 3D shapes used to represent them are shown in Figure 2.\n\nBioPAX may describe the interaction between the components of a pathway but it does not define layout co-ordinates for visualisations, even if the original source of the information, such as Reactome, contained this information. In the absence of layout information, a graph layout must be computed algorithmically. We recommend the Fast Multipole Multilevel Method (FMMM) layout algorithm, implemented within BioLayout, for use with BioPAX networks29. FMMM is a force-directed layout algorithm, introduced in BioLayout version 3.1 that allows graph layout to be computed highly efficiently, with a small number of iterations. The algorithm produces elegantly laid out graphs (Figure 3A) in both 2D and 3D, with sparsely arranged nodes and is particularly useful for the visualisation of large structured networks, such as those obtained from BioPAX.\n\n(A) 3D network view of a BioPAX query. Different entity/concept types defined in BioPAX Level 3 are translated into nodes of different size, shape and colour depending on type. Graphs may be explored, edited and names displayed. Options for these functionalities are contained within the BioLayout menus. (B) The names and node types of selected nodes may be viewed with the Class Viewer, which also provides information on the number of edges. Within this window nodes may be selected/deselected, searched by name or class and information exported.\n\n\nExploration of networks\n\nBioLayout’s Class Viewer23 is used as an inspector for the graph. The Class Viewer enables a graph to be sub-categorized into classes, based on node type. The classes taken together form a Class Set. During the graph construction process, a Class Set is created for BioPAX features and as the graph nodes are created, each node is assigned to a class with the name of the BioPAX entity type to which it corresponds (Figure 3B).\n\nThe Import Network search dialog may be opened from within the Class Viewer, while navigating a gene co-expression network, using the Search Database function. This opens the dialog with the Keywords field pre-populated with gene names from selected nodes within the graph, enabling the user to search for pathways that involve the genes of interest. This represents a means of directly integrating genomic data with pathway data. Similarly, an analysis of a gene expression network or similar, e.g. clustering of co-expression modules, may be exported (File -> Export -> Class Sets As File) and then, assuming that the node identifiers are the same between the two networks, imported (File -> Import -> Class Sets…) whilst visualising a BioPAX network. In this way, the genes of interest in the expression network can be highlighted on the interaction network or vice versa.\n\n\nConclusion\n\nThere is now a wealth of pathway and protein interaction data in the public domain, collected and curated at great expense. However, accessing and using these data has proved challenging for many due to the lack of standard formats for data exchange between resources. The BioPAX standard has gone a long way to resolve this issue and has been widely adopted by the community. The Pathway Commons database has therefore been able to amalgamate the information stored in a number of the main pathway/interaction resources, making the information available through the CPath2 web service. Here we report our implementation of data query and import functionality within BioLayout Express3D version 3.2, thereby leveraging a powerful tool to support the visualisation and analysis of large pathway and protein interaction networks. The data stored in the Pathway Commons resource may now be easily searched and hits combined. The resulting networks can be displayed in 2D or 3D using a graphical display language that differentiates between the entity types described in the BioPAX hierarchy. Within the tool, the graphs can be explored and edited and where necessary exported for visualisation within other tools.\n\n\nSoftware availability\n\nSoftware and source code are available from http://www.biolayout.org.\n\nhttp://dx.doi.org/10.5281/zenodo.1221630\n\nGNU Public Licence version 3 http://www.gnu.org/copyleft/gpl.html", "appendix": "Author contributions\n\n\n\nD.W.W. and T.A. implemented the software described in this paper and A.J.E. advised and assisted with the implementation. T.C.F. oversaw the work described here and co-authored the paper with D.W.W.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nT.C.F. is funded by an Institute Strategic Grant from the Biotechnology and Biological Sciences Research Council (BBSRC) [grant number BB/JO1446X/1] and development of BioLayout Express3D is funded by BBSRC grant BB/I001107/1.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe would like to thank Igor Rodchenkov, Emek Demir and Gary Bader for valuable support with implementing Pathway Commons and PaxTools within BioLayout Express3D.\n\n\nReferences\n\nCroft D, Mundo AF, Haw R, et al.: The Reactome pathway knowledgebase. Nucleic Acids Res. 2014; 42(Database issue): D472–7. 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Application of Graph Layout Algorithms for the Visualization of Biological Networks in 3D. Graph Drawing: Springer. Reference Source\n\nWright DW, Angus T, Enright AJ, et al.: BioLayout Express3D Version 3.2. Zenodo. 2014. Data Source" }	[ { "id": "6455", "date": "27 Oct 2014", "name": "Gary D Bader", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe BioPAX loading and visualization support for the established BioLayout Express 3D network visualization software. As far as I know, this is the only software capable of 3D display of BioPAX derived networks. The new feature seems to work very well as described. I have a few minor comments to improve the manuscript. Minor notes:“the persistent Uniform Resource Identifier (URI)” – URIs in Pathway Commons are not guaranteed to be consistent between releases. They can be persistent if they are externally standardized e.g. uniprot IDs, but internal pathway commons URIs may change from release to release. The manuscript would benefit from one biological use case. The highlighting expression data paragraph is interesting, but including a biological example would help potential biologist users know how to use the software for a particular analysis. It would be nice to see a future directions section e.g. is there a plan to visualize SIF networks derived from BioPAX? It would be useful to briefly mention some examples of information that is not covered in the BioPAX to mEPN mapping e.g. post-translational modifications on proteins, the directionality of edges e.g. that could distinguish between left and right participants of an interaction vs. all participants of an interaction. Some of this information is possible to show in other visualization options, such as the Systems Biology Graphical Notation, which the authors may want to reference. Software comments:The top pathways feature requires entering a keyword before the Search button is active, but with this query, it seems that a keyword is not necessary, as any query always shows all top pathways. A number of nodes have ‘null’ labels e.g. if they are interaction nodes. It would be nice to be able to suppress these labels somehow. It would be useful to see other information associated with the nodes e.g. data source, links to external databases. Some of this information is already shown in section 6 in panel A of the import network dialog shown in Figure 1. Is it possible to choose another layout after a network has been loaded? Bug: Clicking the ‘i’ information button on the right hand button tool bar brings up a help dialog called the ‘navigation wizard’. The links to PDFs at the bottom of this screen go to non-existent web pages. The edge names are all ‘0’, but could be the type of edge. Minor text fixes:“within BioLayout, the user may refine searches by keywords, species, data source and BioPAX type” -> BioPAX class type.", "responses": [] }, { "id": "8200", "date": "07 Apr 2015", "name": "Jianguo Xia", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral Comments: The BioLayout Express 3D is a very useful tool, especially its unique 3D rendering capability. The supports for importing network files in BioPAX level 3 format and integration with Pathway Commons appear to work well as described in the paper. My comments are mainly concerned with network visualization.Navigation in large/dense networks: Users often want to zoom in to a particular node/region of the network to view the connections and structures. It is difficult to achieve this using right-mouse button. A better approach may be to add support for three-wheel mouse – point the mouse to the node and then scroll to zoom in to the pointer direction. Enhancing support for node search in a dense network:Users should be able to click to highlight AND zoom in to it. Better scaling control (esp. for 2D view):Users would expect that node overlapping issue would be resolved by zooming in to the region. This is not the case during testing. Rather than simply getting bigger in size, both the sizes and relatives distance between nodes should be scaled properly when users zoom in. If there are some advanced options to achieve this effect, the default parameters should be improved. Bugs (Version 3.3, Mac version)On the 2D view, select and drag a node to a new position, the mouse pointer and the node are not well synchronized during the process. Black screen also occurs sometimes. Additional comments / wish list:In addition to comparing their abilities in supporting BioPAX format, a general discussion on network visualization features of these popular tools will be very useful for new users to make informed choices. The standalone tools have the advantage for dealing with large data. The current tool can be improved with regard to more layout options and better performance for large networks. Given the increasing popularity for web-based visualization, the support for exporting the graph for D3.js rendering is certainly a welcome step. An online version for 3D network visualization (maybe based on WebGL?) will be highly received by the community.", "responses": [] } ]	1	https://f1000research.com/articles/3-246
https://f1000research.com/articles/3-245/v1	16 Oct 14	{ "type": "Research Article", "title": "Late cardiac sodium current can be assessed using automated patch-clamp", "authors": [ "Morgan Chevalier", "Bogdan Amuzescu", "Vaibhavkumar Gawali", "Hannes Todt", "Thomas Knott", "Olaf Scheel", "Hugues Abriel", "Morgan Chevalier", "Bogdan Amuzescu", "Vaibhavkumar Gawali", "Hannes Todt", "Thomas Knott", "Olaf Scheel" ], "abstract": "The cardiac late Na+ current is generated by a small fraction of voltage-dependent Na+ channels that undergo a conformational change to a burst-gating mode, with repeated openings and closures during the action potential (AP) plateau. Its magnitude can be augmented by inactivation-defective mutations, myocardial ischemia, or prolonged exposure to chemical compounds leading to drug-induced (di)-long QT syndrome, and results in an increased susceptibility to cardiac arrhythmias. Using CytoPatch™ 2 automated patch-clamp equipment, we performed whole-cell recordings in HEK293 cells stably expressing human Nav1.5, and measured the late Na+ component as average current over the last 100 ms of 300 ms depolarizing pulses to -10 mV from a holding potential of -100 mV, with a repetition frequency of 0.33 Hz. Averaged values in different steady-state experimental conditions were further corrected by the subtraction of current average during the application of tetrodotoxin (TTX) 30 μM. We show that ranolazine at 10 and 30 μM in 3 min applications reduced the late Na+ current to 75.0 ± 2.7% (mean ± SEM, n = 17) and 58.4 ± 3.5% (n = 18) of initial levels, respectively, while a 5 min application of veratridine 1 μM resulted in a reversible current increase to 269.1 ± 16.1% (n = 28) of initial values. Using fluctuation analysis, we observed that ranolazine 30 μM decreased mean open probability p from 0.6 to 0.38 without modifying the number of active channels n, while veratridine 1 μM increased n 2.5-fold without changing p. In human iPSC-derived cardiomyocytes, veratridine 1 μM reversibly increased APD90 2.12 ± 0.41-fold (mean ± SEM, n = 6). This effect is attributable to inactivation removal in Nav1.5 channels, since significant inhibitory effects on hERG current were detected at higher concentrations in hERG-expressing HEK293 cells, with a 28.9 ± 6.0% inhibition (mean ± SD, n = 10) with 50 μM veratridine.", "keywords": [ "late Na+ current", "veratridine", "ranolazine", "tetrodotoxin", "automated patch-clamp", "action potential", "Nav1.5", "hERG", "iPSC-derived cardiomyocyte", "HEK293" ], "content": "Introduction\n\nThe late cardiac Na+ current can be recorded 10–100 milliseconds after membrane depolarization as a sustained inward current component (Isus) in cardiomyocytes1. This late current represents a fraction of voltage-dependent Na+ channels that fail to inactivate after the initial opening. Instead, these channels change to a conformation with frequent late re-openings in a so-called burst mode2. Among other mechanisms, calmodulin kinase II overexpression in chronic heart failure increases the rate of transition to the late bursting mode conformation2. The phenomenon is also observed in Nav1.5 inactivation-deficient mutants, such as ΔKPQ3 or 1795InsD4,5, leading to a specific form of congenital long QT syndrome, LQT-36–8.\n\nGiven its role in cardiac arrhythmogenesis9,10, pharmacological inhibition of the late Na+ current component (INa late) is seen as an anti-arrhythmic strategy. Novel late sodium channel blockers, such as the partial fatty acid beta-oxidation inhibitor, ranolazine11–15, are used for both myocardial ischemia and to alleviate neuropathic pain and show a preferential affinity for the burst mode conformation of Na+ channels16–21. Other compounds, such as veratridine, a steroid-derived alkaloid extracted from rhizomes of Veratrum album or seeds of Schoenocaulon officinale22,23, preferentially bind to activated Na+ channels, impeding inactivation and leading to increased nerve excitability.\n\nThe objectives of this study were to (1) record the veratridine-dependent increase in the late Na+ current using a HEK293 cell line stably transfected with human Nav1.524, (2) measure the ranolazine-induced inhibition of basal and veratridine-activated INa late, and (3) asses the effects of veratridine at the same concentrations on action potentials (APs) in hiPSC-cardiomyocyte preparations externally paced in current-clamp mode. In parallel we also tested the inhibitory effects of veratridine on hERG1 current in stably transfected HEK293 cells. All these experiments were performed using CytoPatch™2 automated patch-clamp equipment.\n\n\nMaterials and methods\n\nAll experiments were performed using the CytoPatch™2, using standard dual-channel Cytocentrics chips with embedded quartz pipette tips 2 μM in diameter. For whole-cell late Na+ current recordings, the voltage-clamp protocol consisted of repeated depolarizing pulses of -10 mV amplitude and 300 ms duration, from a holding potential of -100 mV, to allow a substantial removal from inactivation of Nav1.5 channels. The peak and late Na+ current were plotted and monitored over the entire duration of experiment, and extracted from recorded data for further analysis. Pharmacological compounds were applied in a predefined sequence using the dispensing needle of automated equipment. All experiments were performed at room temperature (21–22°C).\n\nFor whole-cell hERG current recordings, cells were held at -70 mV. After a brief 100-ms prepulse to -50 mV to determine the current leak, a 2-s depolarizing voltage step to +40 mV was followed by a 2-s step to -50 mV to elicit hERG tail currents every 10 s. Peak tail current amplitude was corrected by the leak current and the corrected peak tail current was averaged over the last three pulses of the control phase (Ictrl) and the application phase (Icpd), respectively. From these averaged values the hERG tail current inhibition was calculated as follows:\n\nInhibition = 1 – (Icpd/Ictrl)\n\niPSC-CM recordings were performed in current-clamp mode, using repeated sweeps consisting of 3 injected current pulses of 2000 pA amplitude, 0.5 ms duration, at 3-s intervals. During solution uptake the system was switched to voltage-clamp mode.\n\nHEK293 cells stably expressing either the human Nav1.5 channel or the hERG K+ channel were used as ready-to-use frozen Instant cells (product of Cytocentrics). Cells, kept in liquid nitrogen, were quickly thawed, centrifuged, resuspended in extracellular solution at a density of ~106 cells/ml, and used for experiments within 3 hours. For other experiments, Nav1.5-expressing cells were cultured in 25 cm2 flasks in DMEM supplemented with 10% fetal bovine serum and 100 μg/ml zeozin, and kept at 37°C, 8% CO2 in a humidified incubator. Adherent cell monolayers were detached with Versene (ethylenediaminotetraacetic acid – EDTA 0.02% in phosphate buffered saline - PBS), centrifuged for 2 min at 100 × g, and resuspended in hERG external solution.\n\nhiPSC-derived iCell® Cardiomyocytes were kindly provided by Cellular Dynamics International (Madison, WI) as frozen samples, and cultured in monolayers in 12-well plates coated with 0.1% gelatin, for up to 41 days. The thawing/plating and maintenance media were provided by the cell supplier. For detachment, the monolayers were rinsed twice with calcium and magnesium-free PBS, then incubated for 2 min with trypsin 0.1% at 37°C. 1.5 ml of medium was added per well, the cells were suspended, centrifuged at 180 × g for 5 min, and resuspended in a 1:1 mixture of culture medium and hERG extracellular solution.\n\nFor recordings in Nav1.5-transfected HEK293 cells the external solution had the following composition (in mM): NaCl 130, CsCl 5, CaCl2 2, MgCl2 1.2, HEPES 10, D-glucose 5, pH 7.4, osmolality 320 mOsm/kg. The internal solution contained (in mM): Cs-aspartate 70, CsCl 60, CaCl2 1, MgCl2 1, Na2ATP 5, EGTA 11, HEPES 10, pH 7.2 with CsOH, osmolality 290 mOsm/kg. For recordings in iPSC-derived cardiomyocytes and hERG-transfected HEK293 cells the extracellular solution had the following composition (in mM): 140 NaCl, 2.5 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, 10 Glucose, 15 Sucrose. The pH was adjusted to 7.4 with NaOH 1M and the osmolality to 320 (± 5) mOsmol/kg with sucrose 1M, and the storage temperature was 4°C. The intracellular solution contained (in mM): 100 K Gluconate, 20 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 11 EGTA-KOH, 4 ATP-Mg2+, 3 Phosphocreatine-Na2-H2O, 9 Sucrose. The pH was adjusted to 7.2 with KOH 1M, the osmolality to 295 (± 5) mOsmol/l, and then it was stored in 10-ml aliquots at -20°C, thawed and used within 4 hours. Veratridine (Sigma V5754) working solutions at 1, 5 and 50 μM were prepared from a 50 mM stock solution in ethanol. Ranolazine dihydrochloride (Sigma R6152) 10 and 30 μM was prepared from a 10 mM stock solution in DMSO. TTX (BN0518, Biotrend, Zurich, CH) was prepared from a 1 mM aqueous stock solution.\n\nThe software files generated during the recordings were stored on computer hard disks. Patch clamp data were analyzed using the CytoPatchTM software and exported to Microsoft Excel or pClamp10 (Axon Instruments, part of Molecular Devices, Sunnyvale, CA) for further analysis, using a proprietary conversion tool. APD90 analysis for current-clamp recordings in iPSC-derived cardiomyocytes was performed with self-written software routines. APD90 was computed as the duration between the point of maximal AP upstroke speed during phase 0 to recovery of 90% of the difference between peak upstroke potential and resting potential. In the case of veratridine application, if recovery was incomplete during the 3-s interstimulus interval, APD90 was computed over several pacing cycles. For statistical analysis, one-way ANOVA for independent samples with Dunnett’s post-hoc comparison, as well as Student’s t tests where appropriate, were applied, at a level of significance of p ≤ 0.05, using the GraphPad Prism software (La Jolla, CA).\n\n\nResults\n\nUsing automated voltage-clamp protocols applied to human Nav1.5-expressing HEK293 cells in the above mentioned conditions, we routinely recorded, in high-quality seal conditions (both seal and membrane resistance > 1 GΩ), and with stability for at least 20 min, whole-cell Na+ currents. These currents were elicited by membrane depolarization to -10 mV from a holding potential of -100 mV, required for the proper removal from inactivation of cardiac Na+ channels. The late Na+ current component (INa late) was automatically computed and plotted as time average over the last 100 ms of the 300-ms depolarizing pulse of each sweep. Figure 1A shows the time course of 5 averaged experiments including repeated applications of veratridine 1 μM with different durations (2 min, 1 min, 5 min), as well as a 1-min application of TTX 30 μM, resulting in the rapid complete block of late Na+ current. The average current level during TTX application in each experiment was subsequently subtracted from all other averaged steady-state levels to obtain unbiased estimations of INa late in different experimental conditions. In general, reversibility was good and rapid upon TTX wash-out.\n\nA. Time course of INa late (average transmembrane current during the last 100 ms of a 300-ms depolarizing pulse to -10 mV from a holding potential of -100 mV) during a typical experiment with repeated applications of veratridine 1 μM and a final application of TTX 30 μM. B. Overlap of individual sweeps showing the peak and late component of INa during the application of veratridine 1 μM, the co-application with ranolazine 10 μM, and with TTX 30 μM. The peak and late INa component are shown separately in inserts using magnified time and voltage scales, respectively.\n\nUnder control conditions, a 5-min application of veratridine 1 μM induced an increase of the late Na+ current by 269.1 ± 16.1% (mean ± SEM, n = 28) of initial values (Table 1 and Figure 2). Figure 1B shows representative current traces of selected sweeps, as well as magnified inserts of the peak and late phase, within an individual experiment. The application of veratridine 1 μM increased the late current amplitude by more than two-fold relative to the TTX 30 μM reference trace, without any noticeable effect on peak current, while the co-application of ranolazine 10 μM reduced the peak current with apparently no effect on the late component. Systematic experiments with ranolazine applied alone demonstrated INa late reduction to 75.0 ± 2.7% (mean ± SEM, n = 17) of initial values at 10 μM, and 58.4 ± 3.5% (mean ± SEM, n = 18) at 30 μM (Table 1 and Figure 2).\n\n*INa late values were computed by subtraction of averaged amplitude in the presence of TTX 30 μM from averaged amplitudes in all other experimental conditions\n\n§ using two-tailed Student’s t test for paired samples\n\nA. Time plots of INa late in four typical experiments. Intervals of application of ranolazine 10 or 30 μM, TTX 30 μM, and veratridine 1 μM are indicated. Right panels: INa late traces from 260 to 270 ms at the time points marked with arrows and numbered in the left panels. B and C. Effects of ranolazine 10 and 30 μM, respectively, on relative levels of TTX-sensitive late Na+ current. D. Effect of veratridine 1 μM. Mean values are indicated for each condition, and error bars represent SEM.\n\nFigure 3 shows the power density spectra of Fourier-transformed traces recorded under different conditions, as numbered in the upper time course of the experiments. Presumably, the opening and closure of Nav1.5 channels in the late gating mode conformation produces a Lorentzian component with corner frequency above 2 KHz. However, due to the reduced number of individual signal sources, the plateau spectral power density was small and difficult to distinguish. A better approach to fluctuation analysis is by the computation of single-channel parameters such as the mean open probability p and the average number of channels n, starting from the well-known estimations of current variance σ2 and macroscopic current I25:\n\nA. Time course of a typical experiment in Nav1.5-expressing cells showing the effects of ranolazine 30 μM, TTX 30 μM, and veratridine 1 μM on INa late. B. Averaged Fourier transforms of multiple traces recorded in different conditions in the experiment shown in A: 1. initial control; 2. ranolazine 30 μM; 3. TTX 30 μM; 4. veratridine 1 μM; 5. veratridine 50 μM. C. Fluctuation analysis of individual traces recorded during the five distinct periods shown in A allowed the distinction of individual channel gating events and the computation of the average open probability p and the average number of channels n for each condition.\n\nσ2 = npi2 – np2i2 = np(1 – p)i2 and I = npi\n\nWe used a unitary Na+ current amplitude i at -10 mV of 1.43 pA, based on the unitary current amplitude of 2 pA at -40 mV observed in single-channel recordings of Nav1.5 late currents in HEK293 cells26 and a reversal potential of +65 mV. In doing so, we could evaluate the mean open probability p and further the average number of Na+ channels n open in the late mode in selected traces, over the last 100 ms of activity during the 300-ms depolarizing pulses. It was observed that 30 μM ranolazine application resulted in a decrease of mean open probability from 0.6 to 0.38, without affecting the average number of channels contributing the late Na+ current (n = 4), in agreement with estimates obtained from averaged macroscopic currents. On the other hand, veratridine 1 μM increased the number of channels in late gating mode to n = 10, without influencing the mean open probability.\n\nTo get a better understanding of the role that the late Na+ current may play in arrhythmogenesis and in the complex mechanisms of di-LQT syndrome, we performed a series of automated patch-clamp experiments on hiPSC-derived cardiomyocytes with current-clamp recordings of stimulus-triggered APs. Each recorded sweep contained responses elicited by three injected current stimuli (0.5 ms duration, 2 nA amplitude). As shown in Figure 4, veratridine 1 μM induced a marked prolongation of AP duration, reaching saturation in less than 3 min. Further application of 5 μM veratridine resulted in further APD90 prolongation, exceeding the interstimulus interval. There was a slow trend to reversibility during the wash-out phase. Quantitative results for veratridine 1 μM application are summarized in Table 2. Thus, in n = 6 experiments at room temperature the relative APD90 prolongation induced by veratridine was 2.12 ± 0.41 fold (mean ± SEM), reversible to 1.21 ± 0.50 fold at wash-out (mean ± SEM, n = 4).\n\nCurrent-clamp AP recordings using a pacing protocol with injected current stimuli of 0.5-ms duration and 2 nA amplitude repeated at 3-s intervals. The overlap of traces in control conditions at the start of the experiment, with 1 μM veratridine (onset and full effect), with 5 μM veratridine, and during wash-out, shows partial recovery.\n\nRelative values are computed taking initial values as reference\n\np values for Student’s t test for paired samples, two-tailed, for condition tested vs. initial values\n\nn = number of cells included in analysis\n\nLast, we performed experiments using HEK293 cells stably expressing hERG1 to assess the effects of veratridine at different concentrations on this current component, in order to better understand the complex effects of this compound on the action potential of iPSC-derived cardiomyocytes. As shown in Figure 5, veratridine concentrations up to 5 μM did not produce levels of hERG inhibition significantly different than the diluting vehicle alone (ethanol 0.1%), while at 50 μM there was a 28.9 ± 6.0% (mean ± SD, n = 10) inhibition of peak hERG current, a statistically significant effect (p < 0.0001, one-way ANOVA for independent samples with Dunnett’s multiple comparison test vs. control).\n\nA. Current traces in control conditions (upper) and during application of veratridine 50 μM (lower trace). B. Percentages of peak hERG current inhibition by different concentrations of veratridine and control ethanol 0.1% vehicle. Error bars represent SD. Number of experiments: control (n = 6), veratridine 0.1 μM (n = 10), 1 μM (n = 5), 5 μM (n = 7), 50 μM (n = 10).\n\n\nDiscussion\n\nThe main findings of the present study using the CytoPatchTM2 instrument are the following: (1) inhibition of cardiac late Na+ current to 75.0% and 58.4% of initial values by ranolazine 10 and 30 μM, respectively; (2) activation of the same current by veratridine 1 μM to 269.1% of initial values; and (3) prolongation of APD90 by veratridine 1 μM to 212% of initial values in human iPSC-derived cardiomyocytes. We have succeeded in recording and analysing the small (a few picoA) late Na+ current component in a human cell line stably transfected with wild-type human SCN5A, using automated patch-clamp technology, with a voltage protocol different from that used in the first electrophysiology characterization of ΔKPQ LQT-3 mutant Na+ channels3. We observed that instrumental noise does not significantly affect the average current value over the last 100 ms of a 300-ms depolarizing pulse from -100 to -10 mV, therefore this value can be computed and plotted in real-time, representing a sensitive indicator for evidencing pharmacological effects in automated patch-clamp assays. Another critical parameter is the frequency of repetition of depolarizing stimuli, because it may reveal use dependency for compounds exerting state-dependent binding effects27. Although in the present set of experiments we kept a fixed value of 0.33 Hz, inclusion of different pacing rates in future variants of the assay may offer supplementary information of pharmacological relevance. An important tool in the precise assessment of INa late levels is the application of TTX 30 μM, which results in an almost complete late Na+ current inhibition, and offers a current reference used for the correction by subtraction of all other values in different experimental conditions. An excellent seal stability and access resistance, such as those offered by the Cytocentrics microfluidic chips28, is another prerequisite for successful late Na+ pharmacology experiments.\n\nDue to the limited amplitude of late Na+ current under basal conditions, we increased it using low concentrations of veratridine, a steroid-derived natural alkaloid known to bind to an intramembrane site of Na+ channel pore subunits and, in doing so, stabilize it in the open conformation29,30, contributing to the INa late. Although veratridine at 1 μM resulted in a consistent 2.5 – 3-fold increase in late Na+ current, compared to initial levels (Table 1 and Figure 1–Figure 3), we would not recommend this procedure for pharmacology assays because of possible interactions between veratridine and test compounds. Although we have not systematically explored this phenomenon, preliminary observations, as shown in Figure 1B, suggest a reduced effectiveness of ranolazine 10 μM when co-applied with veratridine, compared to application of ranolazine alone at the same concentration. The value of relative inhibition of the late Na+ current by ranolazine 10 μM in the single compound application obtained in our study (75.0% of control levels) is larger than those obtained with the same concentration of ranolazine on late Na+ current activated by veratridine 40 μM (89% and 81% for holding potentials of -110 mV and -90 mV, respectively)31. An elegant method that may offer pharmacologically relevant information concerning the site and mechanism of action of a certain compound is fluctuation analysis. As shown in Figure 3, when applied to selected traces, this method allows the computation of the mean open probability p and the average number of open channels n contributing to current variance. From this analysis, we hypothesize that ranolazine acts as an open pore blocker, reducing the mean open probability at 30 μM to 63% of initial values, from 0.6 to 0.38, without lowering the number of active channels, while veratridine 1 μM increases the number of active channels in late gating mode 2.5-fold, without influencing their mean open probability.\n\nIn agreement with the significant activatory effects of veratridine 1 μM on cardiac Nav1.5 channels in late gating mode in stably transfected cell lines, we found an important prolongation of action potential duration when the drug was applied to human iPSC-derived cardiomyocytes in current-clamp configuration, using CytoPatch™2 automated patch-clamp equipment. The APD90 was increased over twofold by veratridine 1 μM, with a good reversibility upon wash-out (Table 2), and an even higher prolongation with 5 μM (Figure 4), although in this case we were unable to accurately compute APD90 values for externally paced APs, due to their overlap with the next stimulus. This effect is consistent with the findings of previous studies, showing APD prolongation in bronchial smooth muscle cells upon veratridine application32, and the enhancement of AP burst generation by veratridine in rat trigeminal sensory neurons33. Furthermore, we have demonstrated that hERG inhibition does not play any role in the observed APD prolongation by veratridine at 1 or 5 μM, since significant hERG inhibition occurred with higher veratridine concentrations (Figure 5). A recent combined in vivo and in vitro study proved that the augmentation of the myocardial late Na+ current during ventricular remodeling induced by pulmonary arterial hypertension in rats was accompanied by significant APD90 increases, and a beneficial effect of repeated ranolazine administration via late Na+ current block was demonstrated with a subsequent alleviation of induced intracellular Ca2+ overload34.\n\nIn conclusion, the present study demonstrates that automated patch-clamp, as implemented by the CytoPatch™2 equipment using our proprietary CYTOCENTERING technology and quartz pipette tips embedded in silicon microfluidic chips28,35, which allow high-quality stable seals, lead to reliable late Na+ current pharmacology recordings, with results at least comparable to those obtained in manual patch-clamp experiments. In addition, automation may prove advantageous given the small amplitude of late Na+ current, which therefore requires a large number of experiments for the accurate assessment of pharmacological effects.\n\n\nData availability\n\nF1000Research: Dataset 1. Experimental data showing the effect of veratridine, ranolazine and TTX on late Na+ currents in cultured cells, 10.5256/f1000research.5544.d3699336\n\n\nAbbreviations\n\nHEK: human embryo kidney\n\nCHO: Chinese hamster ovary\n\nhERG: human ether-á-go-go related gene\n\nSEM: standard error of the means\n\nSD: standard deviation\n\nDMEM: Dulbecco’s modified Eagle’s medium\n\nDMSO: dimethylsulfoxide\n\nHEPES: N-2-hydroxyethylpiperazine-N´-2-ethansulfonic acid\n\nEGTA: ethylene glycol-bis(ß-aminoethyl ether)-N,N,N,N´-tetraacetic acid\n\nATP: adenosine triphosphate\n\nTTX: tetrodotoxin\n\nIC: intracellular solution (pipette-filling solution)\n\nEC: extracellular solution\n\nPBS: phosphate-buffered saline\n\niPSC-CM: induced pluripotent stem cell-derived cardiomyocytes\n\nAPD: action potential duration", "appendix": "Author contributions\n\n\n\nHA and TK conceived the study. OS and BA designed the experiments. MC, BA, VG and OS carried out the research. OS and BA prepared the first draft of the manuscript. HT and HA contributed to the experimental design and preparation of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNone of the authors has competing interests concerning the present study, except for the participation of TK, OS and BA in the Cytocentrics company.\n\n\nGrant information\n\nThis study was in part funded by a BMBF grant 03WKCC4D, TransCure NCCR Network to HA, and Austrian Science Fund FWF (grant W1232-B11).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors gratefully acknowledge Stefanie Frech, Jörg Eisfeld, Ingrid Rosenkranz, Juliane Böttcher, Christian Scherpeltz, Yassine M. Amarcouch and Jean-Sébastien Rougier for their support, and Cellular Dynamics International for the provision of iCell® Cardiomyocytes.\n\n\nReferences\n\nClancy CE, Tateyama M, Liu H, et al.: Non-equilibrium gating in cardiac Na+ channels: an original mechanism of arrhythmia. Circulation. 2003; 107(17): 2233–7. PubMed Abstract \| Publisher Full Text\n\nGrandi E, Puglisi JL, Wagner S, et al.: Simulation of Ca-calmodulin-dependent protein kinase II on rabbit ventricular myocyte ion currents and action potentials. Biophys J. 2007; 93(11): 3835–47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBennett PB, Yazawa K, Makita N, et al.: Molecular mechanism for an inherited cardiac arrhythmia. Nature. 1995; 376(6542): 683–5. PubMed Abstract \| Publisher Full Text\n\nBezzina C, Veldkamp MW, van Den Berg MP, et al.: A single Na(+) channel mutation causing both long-QT and Brugada syndromes. Circ Res. 1999; 85(12): 1206–13. 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Heart Rhythm. 2009; 6(11): 1625–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStett A, Burkhardt C, Weber U, et al.: CYTOCENTERING: a novel technique enabling automated cell-by-cell-patch clamping with the CYTOPATCH chip. Recept Chann. 2003; 9(1): 59–66. PubMed Abstract\n\nCatterall WA: Cooperative activation of action potential Na+ ionophore by neurotoxins. Proc Natl Acad Sci U S A. 1975; 72(5): 1782–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUlbricht W: Effects of veratridine on sodium currents and fluxes. Rev Physiol Biochem Pharmacol. 1998; 133: 1–54. PubMed Abstract \| Publisher Full Text\n\nLe Grand B, Pignier C, Letienne R, et al.: Sodium late current blockers in ischemia reperfusion: is the bullet magic? J Med Chem. 2008; 51(13): 3856–66. PubMed Abstract \| Publisher Full Text\n\nBradley E, Webb TI, Hollywood MA, et al.: The cardiac sodium current Na(v)1.5 is functionally expressed in rabbit bronchial smooth muscle cells. Am J Physiol Cell Physiol. 2013; 305(4): C427–35. PubMed Abstract \| Publisher Full Text\n\nTsuruyama K, Hsiao CF, Chandler SH: Participation of a persistent sodium current and calcium-activated nonspecific cationic current to burst generation in trigeminal principal sensory neurons. J Neurophysiol. 2013; 110(8): 1903–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRocchetti M, Sala L, Rizzetto R, et al.: Ranolazine prevents INaL enhancement and blunts myocardial remodelling in a model of pulmonary hypertension. Cardiovasc Res. 2014; 104(1): 37–48. PubMed Abstract \| Publisher Full Text\n\nScheel O, Himmel H, Rascher-Eggstein G, et al.: Introduction of a modular automated voltage-clamp platform and its correlation with manual human ether-à-go-go related gene voltage-clamp data. Assay Drug Dev Technol. 2011; 9(6): 600–7. PubMed Abstract \| Publisher Full Text\n\nChevalier M, Amuzescu B, Gawali V, et al.: Experimental data showing the effect of veratridine, ranolazine and TTX on late Na+ currents in cultured cells. F1000Research. 2014. Data Source" }	[ { "id": "6442", "date": "28 Oct 2014", "name": "Eva Delpon", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper by Chevalier et al. analyzed whether late sodium current (INaL) can be assessed using an automated patch-clamp device. To this end, the INaL effects of ranolazine (a well known INaL inhibitor) and veratridine (an INaL activator) were described. The authors tested the CytoPatch automated patch-clamp equipment and performed whole-cell recordings in HEK293 cells stably transfected with human Nav1.5. Furthermore, they also tested the electrophysiological properties of human induced pluripotent stem cell-derived cardiomyocytes (hiPS) provided by Cellular Dynamics International. The title and abstract are appropriate for the content of the text. Furthermore, the article is well constructed, the experiments were well conducted, and analysis was well performed.INaL is a small current component generated by a fraction of Nav1.5 channels that instead to entering in the inactivated state, rapidly reopened in a burst mode. INaL critically determines action potential duration (APD), in such a way that both acquired (myocardial ischemia and heart failure among others) or inherited (long QT type 3) diseases that augmented the INaL magnitude also increase the susceptibility to cardiac arrhythmias. Therefore, INaL has been recognized as an important target for the development of drugs with either antiischemic or antiarrhythmic effects. Unfortunately, accurate measurement of INaL is a time consuming and technical challenge because of its extra-small density. The automated patch clamp device tested by Chevalier et al. resolves this problem and allows fast and reliable INaL measurements.The results here presented merit some comments and arise some unresolved questions. First, in some experiments (such is the case in experiments B and D in Figure 2) current recordings obtained before the ranolazine perfusion seem to be quite unstable. Indeed, the amplitude progressively increased to a maximum value that was considered as the control value (highlighted with arrows). Can this problem be overcome? Is this a consequence of a slow intracellular dialysis? Is it a consequence of a time-dependent shift of the voltage dependence of activation/inactivation? Second, as shown in Figure 2, intensity of drug effects seems to be quite variable. In fact, experiments A, B, C, and D in Figure 2 and panel 2D, demonstrated that veratridine augmentation ranged from 0-400%. Even assuming the normal biological variability, we wonder as to whether this broad range of effect intensities can be justified by changes in the perfusion system. Has been the automated dispensing system tested? If not, we suggest testing the effects of several K+ concentrations on inward rectifier currents generated by Kir2.1 channels (IKir2.1).The authors demonstrated that the recording quality was so high that the automated device allows to the differentiation between noise and current, even when measuring currents of less than 5 pA of amplitude. In order to make more precise mechanistic assumptions, the authors performed an elegant estimation of current variance (σ2) and macroscopic current (I) following the procedure described more than 30 years ago by Van Driessche and Lindemann 1. By means of this method, Chevalier et al. concluded that ranolazine acts as an open pore blocker reducing the open channel probability, while veratridine increases the number of channels in the burst mode. We respectfully would like to stress that these considerations must be put in context from a pharmacological point of view. We do not doubt that ranolazine acts as an open channel blocker, what it seems clear however, is that its onset block kinetics has to be “ultra” slow, otherwise ranolazine would decrease peak INaL even at low frequencies of stimulation. This comment points towards the fact that for a precise mechanistic study of ionic current modifying drugs it is mandatory to analyze drug effects with much more complicated pulse protocols. Questions thus are: does this automated equipment allow to the analysis of the frequency-, time-, and voltage-dependent effects of drugs? Can versatile and complicated pulse protocols be applied? Does it allow to a good voltage control even when generated currents are big and fast? If this is not possible, and by means of its extraordinary discrimination between current and noise, this automated patch-clamp equipment will only be helpful for rapid INaL-modifying drug screening. Obviously it will also be perfect to test HERG blocking drug effects as demanded by the regulatory authorities.Finally, as cardiac electrophysiologists, we would like to stress that it seems that our dream of testing drug effects on human ventricular myocytes seems to come true. Indeed, human atrial myocytes are technically, ethically and logistically difficult to get, but human ventricular are almost impossible to be obtained unless from the explanted hearts from patients at the end stage of cardiac diseases. Here the authors demonstrated that ventricular myocytes derived from hiPS generate beautiful action potentials that can be recorded with this automated equipment. The traces shown suggested that there was not alternation in the action potential duration. Is this a consistent finding? How long do last these stable recordings? The only comment is that resting membrane potential seems to be somewhat variable. Can this be resolved? Is it an unexpected veratridine effect? Standardization of maturation methods of ventricular myocytes derived from hiPS will be a big achievement for cardiac cellular electrophysiology which was obliged for years to the imprecise extrapolation of data obtained from a combination of several species none of which was representative of human electrophysiology. The big deal will be the maturation of human atrial myocytes derived from hiPS that fulfil the known characteristics of human atrial cells.Minor points:We suggest suppressing the initial sentence of section 3. We surmise that results obtained from the experiments described in this section cannot serve to understand the role of INaL in arrhythmogenesis.", "responses": [] }, { "id": "6444", "date": "29 Oct 2014", "name": "Céline Fiset", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study, Chevalier et al. had succeeded to record and analyse the late Na+ current (INaL) in HEK293 cells stably transfected with wild-type human SCN5A, using automated patch-clamp technology. They were able to accurately assess the effects of pharmacological agents on INaL. They showed that the cardiac INaL can be inhibited by ranolazine and activated by veratridine. Using fluctuation analysis, they hypothesized that ranolazine would act as an open pore blocker, reducing the open probability without affecting the number of active channels, while veratridine would increase the number of active channels without influencing their open probability. In addition, they also provided additional functional data by performing a series of automated patch-clamp experiments showing that application of veratridine concentrations that increased INaL but had no effects on HERG current prolonged the action potential duration of human iPSC-derived cardiomyocytes. This is an interesting and well-designed study that reports high quality data. Considering the role of late sodium current in the action potential duration, mainly under pathological conditions, and the difficulty to accurately record this small ionic current, the subject of this study is highly relevant. The demonstration of the suitability of the automated patch clamp technology to accurately measure the late sodium current and to assess the effects of pharmacological agents on this current is helpful. In addition, the use of the hiPSC –derived cardiomyoyctes with this approach is also interesting. I do not have any major issues to address. However, the paper could be improved by attention to the following points. All experiments were performed at room temperature. Considering the limited amplitude of the late Na+ current under basal conditions, it could have been useful to perform these experiments at a more physiological temperature to help increase the amplitude of the current. In Figure 1B, it would have been interesting to also present the recordings with ranolaxine alone. The action potential was elicited by the injection of 2 nA of current. This is a rather large current and is probably not just-suprathreshold depolarizing current. Do the hiPSC-derived cardiomyocytes usually require such a high current injection to elicit action potential? For the HERG recordings, is there any specific reason why the external concentration of KCl was only 2.5 mM? Data reported in Table 1 could have been normalized to the cell capacitance to report the current density instead of the current amplitude. For some statistical analysis, an ANOVA would have been more appropriate that a Student’s t tests as several groups were examined (eg., Tables 1 and 2). There are few typographic errors in the Abstract: the units for the drugs ranolaxine, veratridine and TTX are all in millimolar concentrations but should be in the micromolar concentrations.Overall, the study is interesting and describes a methodological approach that could help study the late sodium current. The experiments were well designed and presented and the subject of this manuscript is of importance to the field.", "responses": [] } ]	1	https://f1000research.com/articles/3-245
https://f1000research.com/articles/3-154/v1	04 Jul 14	{ "type": "Research Article", "title": "False memory susceptibility is correlated with categorisation ability in humans", "authors": [ "Kathryn Hunt", "Lars Chittka" ], "abstract": "Our memory is often surprisingly inaccurate, with errors ranging from misremembering minor details of events to generating illusory memories of entire episodes. The pervasiveness of such false memories generates a puzzle: in the face of selection pressure for accuracy of memory, how could such systematic failures have persisted over evolutionary time? It is possible that memory errors are an inevitable by-product of our adaptive memories and that semantic false memories are specifically connected to our ability to learn rules and concepts and to classify objects by category memberships. Here we test this possibility using a standard experimental false memory paradigm and inter-individual variation in verbal categorisation ability. Indeed it turns out that the error scores are significantly negatively correlated, with those individuals scoring fewer errors on the categorisation test being more susceptible to false memory intrusions in a free recall test. A similar trend, though not significant, was observed between individual categorisation ability and false memory susceptibility in a word recognition task. Our results therefore indicate that false memories, to some extent, might be a by-product of our ability to learn rules, categories and concepts.", "keywords": [ "children", "eyes", "false memories", "intelligence", "semantics" ], "content": "Introduction\n\nWhen remembering the past, we typically feel that our memory allows retrieval of events as they really occurred. Yet a large body of work shows that memory is often surprisingly inaccurate, with errors ranging from misremembering minor details of events to generating illusory memories of entire episodes (Loftus, 1997). False memory, the phenomenon of remembering something that actually never occurred, has become a widely studied topic since its origins in Binet’s (1900) La Suggestibilité and Bartlett’s (1932) Remembering. The pervasiveness of such false memories generates an evolutionary puzzle; in the face of selection pressure for accuracy of memory (Dukas, 1999; Mery, 2013; Raine & Chittka, 2008), how could such systematic failures have persisted over evolutionary time? As with perceptual illusions, false memories might be inevitable by-products of otherwise adaptive cognitive processes. Here we explore whether individuals with a higher propensity to form false memories are better at other cognitive tasks, thus generating a trade-off by which certain cognitive capacities (in this case forming links between distinct memories, as in categorisation) cannot be achieved without the cost of memory inaccuracies.\n\nA plethora of experimental paradigms exist for eliciting differing types of false memories in declarative memory, i.e. people’s conscious memory for facts (Brainerd & Reyna, 2005). Episodic (and as such autobiographical) false memories are commonly elicited using the misinformation paradigm, in which information provided or questions asked after an event can bias memory (Loftus, 2005). Conversely, semantic false memories can be elicited using the presentation of lists of semantically related words (Deese, 1959; Roediger & McDermott, 1995). The so called Deese-Roediger-McDermott (DRM) paradigm has become widely used for exploring the malleability of memory. In this paradigm, participants begin by studying lists of words; for example a list may comprise the words mad, fear, hate, rage, temper, fury, ire, wrath, happy, fight, hatred, mean, calm, emotion, enrage. Each list is composed of the 15 strongest associates of one critically non-presented word, for example anger for the above list. Upon free recall of the lists or during a recognition test, the non-presented words are ‘remembered’ at high rates and with high levels of confidence. This high proportion of false memories is attributed to the strength of the associations between the words presented in the lists and the words falsely remembered (Deese, 1959).\n\nWhile such tests might be viewed as rather remote from real-life situations in which the accuracy of memory matters, including episodic memories (DePrince et al., 2004; Freyd & Gleaves, 1996), it has recently been proposed that different types of false memories may share the same underlying mechanisms (Otgaar et al., 2012). These authors showed that children who generate a rich false memory when subjected to a typical false memory implantation paradigm, such as being led to believe they once took a ride in a hot air balloon (which in fact never occurred), are also more susceptible to false memories in a DRM test than children who do not develop a rich implanted false memory. Thus the DRM paradigm, artificial though it may seem, is a useful laboratory paradigm to test individual false memory susceptibility more generally.\n\nClearly false memories cannot in themselves be useful, but like other memory inaccuracies (such as forgetting) they might be by-products of the otherwise adaptive nature of memory processes (Schacter, 1999; Schacter & Dodson, 2001; Schacter et al., 2011). But what cognitive processes might facilitate the generation of false memories as a by-product? It is possible that our abilities for rule learning, association and categorisation might come at a cost when it comes to memorising isolated facts, events, or indeed words. Specifically with respect to the semantic false memories tested in the DRM paradigm, errors might be produced by the ability of individuals to group words together, placing them in categories based on rules for membership. It therefore seems plausible that the creation of these semantic false memories may be a by-product of our ability to group words into categories.\n\nCategorising items is known to generate adaptive benefits such as the ability to learn information more quickly and to show greater efficiency during decision-making (Merritt et al., 2010), but McClelland (1995) argues that whilst such categorisation “is central to our ability to act intelligently” it however “gives rise to distortion as an inherent by-product” (p. 84). It is therefore possible that memory errors are an inevitable fluke of a powerful, adaptive cognitive phenomenon, in the case of semantic false memories our ability to learn rules and concepts, and to classify novel objects by category memberships (Carey, 2011; Chittka & Jensen, 2011). Indeed, categorisation is a strategy to economise on memory, since it allows recognising objects by a limited set of features that define the category, rather than memorising every single possible member of the category (Avarguès-Weber et al., 2011; Chittka & Niven, 2009; Srinivasan, 2006).\n\nOne possibility to explore the potential trade-off between categorisation ability and false memory susceptibility is to exploit variation between individuals, and to test whether superior performance on the one test comes with increased error rates on the other. Inter-individual variation is the raw material for evolution, and offers the possibility to quantify the fitness benefits of cognitive traits in natural settings (Cole et al., 2012; Raine & Chittka, 2008; Rowe & Healy, 2014; Thornton et al., 2014) and to test potential trade-offs between one cognitive capacity and another (Boogert et al., 2011; Raine & Chittka, 2012). Here we investigate a potential correlation between an individual’s proneness to semantic type false memories and their categorisation ability. For this purpose we subjected participants to a DRM paradigm to assess their semantic false memory susceptibility and a test consisting of verbal reasoning questions to assess their ability to form categories. Our findings indicate that false memories, to some extent, might be a by-product of our ability to learn rules, categories and concepts.\n\n\nMethods\n\nThe general method for eliciting false memories was based on Roediger & McDermott (1995) and Stadler et al., (1999). The protocol for the visual presentation of the wordlists was adapted from Peters et al., (2008). The categorisation test was constructed from educational aids published by Coordination Group Publications Ltd (Parsons, 2002a; Parsons, 2002b), Chukra Ltd (2007) and Eleven Plus Exam Group (2010).\n\nThirty-nine 2nd year undergraduate students from the School of Biological & Chemical Sciences, Queen Mary University of London participated in the study. The participants were one full class undertaking a ‘statistics’ module and as such the experiment formed part of their learning, with a report writing task set from the results. Participant demographics were as follows: seven male, thirty-two female, aged nineteen to thirty years. Full ethics approval was obtained from Queen Mary University of London Research Ethics Committee (Ref #0355) and all participants gave written consent of their acceptance to participate in the study.\n\nTo elicit the false memories, eighteen wordlists were used. Each wordlist consisted of the fifteen most commonly associated words of a critical non-presented word. For example the list mad, fear, hate, rage, temper, fury, ire, wrath, happy, fight, hatred, mean, calm, emotion, enrage is composed of the fifteen strongest associates of the word anger and whilst the fifteen words in the list were shown to participants, the critical word anger was not.\n\nThe wordlists were constructed using the first fifteen words listed in the Russell & Jenkins (1954) norms for the critical non-presented words (see Roediger & McDermott, 1995; Stadler et al., 1999 for full details of list construction). The eighteen wordlists were chosen for their known ability to elicit a high proportion of false memories during recall (Stadler et al., 1999). The eighteen critical non-presented words used (and their corresponding fifteen wordlists) were: window, sleep, smell, doctor, sweet, chair, smoke, rough, needle, anger, trash, soft, city, cup, cold, mountain, slow, river (Stadler et al., 1999).\n\nThe wordlists were put into an automated computerised visual presentation (Microsoft Powerpoint 2007, version 12.0.6654.5000) in which each word was displayed in bold, black ‘Calibri Headings’ typeface, font size eighteen. Each word was displayed in the centre of a white screen at a rate of one second per word, with an inter-word interval of approximately five hundred milliseconds. To mark the start and end of a wordlist a white screen containing a black cross was displayed for one second. Following the end of each wordlist a blank white screen was displayed for two minutes. This coincided with the two minute free recall period (see below). The list order was randomised and the words within each list were presented in order of their associative strength to the critical non-presented word, strongest to weakest.\n\nThe recognition test was comprised of one hundred and eight words randomly ordered in four columns of twenty-seven on a sheet of paper. The one hundred and eight words were those from serial positions one, eight, and ten of each of the eighteen studied lists, the eighteen critical lures, and thirty-six unrelated words not found in any of the eighteen lists. The thirty-six unrelated words were selected from the other eighteen word lists published in Stadler et al., (1999) and from the Oxford English Dictionary. The 36 'incorrect' words were: young, chess, circus, march, ink, rye, keys, chequered, soccer, basket, noon, muscle, piano, scribble, bounce, button, feelers, jail, jubilee, rubric, folder, paint, postcard, fan, lamp, book, computer, first, thought, tile, hide, worth, planet, radio, arm, basement.\n\nThe categorisation test consisted of forty-five printed questions. Each question consisted of five words, three of which were associated with one another and two of which were not. Participants were required to circle the two words that were not associated. An example of a question is as follows: 1. curve, arc, crouch, bend, medicine, where curve, arc and bend are the three words associated with one another and crouch and medicine are the words to be correctly circled. Source materials for the categorisation test were example verbal reasoning questions for UK 11+ exams (secondary school entry exams). Questions were reproduced with copyright permission from Coordination Group Publications Ltd (Parsons, 2002a; Parsons, 2002b), Chukra Ltd (2007) and Eleven Plus Exam Group (2010).\n\nAll participants were tested in one sitting. Participants were advised that they would be tested on their memory for lists of words and that they would be required to solve some word puzzles.\n\nParticipants viewed the visual presentation containing the eighteen wordlists on a large screen (240cm width, 180cm height). At the end of each list a two minute recall period was given. During these free recall periods, participants were instructed to write down as many of the words from the list they had just seen as they could remember. Participants were instructed not to guess, but to only write down words that they were reasonably sure they had seen. Participants were provided with a booklet in which to write down their responses.\n\nParticipants then undertook the recognition test. They were instructed to carefully read the words on the sheet provided and to circle any words that they remembered being presented in the eighteen wordlists. Again participants were instructed not to guess but to only circle words they were reasonably sure they had seen.\n\nAfter the final recall period a ten minute break was given, but participants were instructed not to talk to each other about the study. Participants were then given seven minutes to work through the categorisation test. Again they were instructed not to guess, but to only answer those questions to whose answer they were reasonably sure of. Upon completion participants were fully de-briefed as to the purpose of the study.\n\nThe number of critical non-presented words recalled (false memories), the number of critical non-presented words recognised (false memories), and the number of errors made on the categorisation test were calculated for each individual. These were also converted to give percentage errors (out of those possible to produce) to display graphically. We tested the data for normality and found that the distribution departed significantly from a normal distribution (Shapiro-Wilk normality test: p<0.001, skewness=1.830, kurtosis=5.094 (leptokurtic distribution). Therefore a non-parametric correlation analysis (Spearman’s rank correlation coefficient) was used to look for a potential link between categorisation ability (categorisation test errors) and false memory susceptibility (recall and recognition errors). Additional correlations were used on subsets of the data to check for any biasing effects of priming, outliers and age. Finally, the numbers of recall, recognition and categorisation errors were compared between males and females using Wilcoxon rank sum tests to look for an effect of gender. All analyses were carried out using R statistical software (v.2.14.1). P values below 0.05 were deemed significant.\n\n\nResults\n\nThere were substantial inter-individual differences in both participants’ verbal categorisation abilities and their scores on a standardised false memory test. Categorisation errors ranged from 7% to 78% in different individuals (Mean 23%, SD 14%), showing that even though the test we had chosen was originally designed for pre-teens, the task was sufficiently challenging for the tested population to capture a large range of inter-individual variation (Figure 1a). It was important to establish this since if all participants had near-perfect scores (or indeed if all had equally poor scores), the test would not have been suitable to correlate individual variation with other assessments of cognitive performance.\n\na) the percentage of errors scored by individuals on the categorisation test, b) the percentage of false memories (out of those possible to elicit) recalled by individuals during the DRM paradigm and c) the percentage of false memories (out of those possible to elicit) recognised by individuals during the DRM paradigm. N=39. All show a spread of inter-individual variation.\n\nVariation in individual false memory scores was likewise extensive. Recall false memory scores ranged from 0% to 78% (Mean 41%, SD 21%) of possible false memories made (Figure 1b). Two individuals did not recall a single critical non-presented word and thus had a score of zero (and 0%) for recall false memories. Conversely three individuals recalled thirteen out of the possible eighteen false memories (and thus scored 72%), and one participant even scored fourteen (78%). Recognition false memory scores ranged from 17% to 94% (Mean 63%, SD 21%) of possible false memories made (Figure 1c). Five individuals recognised five or less of the critical non-presented words (and thus scored 28% or less), whilst eighteen individuals recognised thirteen or more out of the eighteen possible false memories (and thus scored 72% or more).\n\nWe found a significant negative correlation between individuals’ categorisation error scores (given as the number of questions answered incorrectly on the categorisation test) and their false memory susceptibility during free recall (given as the number of critical non-presented words recalled) (rs=-0.345, df=37, p=0.032, Figure 2), thus those individuals scoring fewer errors on the categorisation test were more susceptible to false memory intrusions during free recall. In other words, participants that performed worse on the one test performed better on the other, and vice versa – indicating an inter-individual trade-off between categorisation ability on the one hand and false memory susceptibility during free recall on the other.\n\nIndividuals’ categorisation abilities (given as the percentage of questions answered incorrectly on the categorisation test) plotted against their susceptibilities to false memories (given as the percentage of critical non-presented words recalled, out of those possible). Those individuals scoring fewer errors on the categorisation test were more susceptible to false memory intrusions and correspondingly had a higher false memory score (rs=-0.345, df=37, p=0.032).\n\nWe also found a slight negative correlation between individuals’ categorisation error scores (given as the number of questions answered incorrectly on the categorisation test) and their false memory susceptibility during recognition (given as the number of critical non-presented words recognised); however this trend was not significant (rs=-0.202, df=37, p=0.219, Figure 3).\n\nTo exclude the possibility that any correlation could be caused by priming, the data were also analysed excluding those categorisation test questions that contained words previously presented in the wordlists, and non-presented as one of the critical non-presented words. In our experiment for example, priming may have meant that the word eye presented as part of a question in the categorisation test: 41. Eye neck nose mouth shoulder, may have been preferentially selected as an answer due to its previous presentation in the word list associated with the critical non-presented word needle – thread, pin, eye, sewing, sharp, point, prick, thimble, haystack, thorn, hurt, injection, syringe, cloth, knitting. As such the scores for twelve questions were removed. A significant negative correlation was still found for free recall and a moderate negative correlation still found for recognition; thus priming cannot account for the result (recall: rs=-0.362, df=37, p=0.024, recognition: rs=-0.206, df=37, p=0.208).\n\nIndividuals’ categorisation abilities (given as the percentage of questions answered incorrectly on the categorisation test) plotted against their susceptibilities to false memories (given as the percentage of critical non-presented words recognised, out of those possible). Again, those individuals scoring fewer errors on the categorisation test were more susceptible to false memory intrusions and correspondingly had a higher false memory score, though in this case the correlation was not significant: rs=-0.202, df=37, p=0.219.\n\nAdditionally, the removal of an outlier (a residuals vs. leverage plot showed a Cook’s distance greater than 0.5 for participant 24, see Dataset 1) did not change the statistical significance of the original result, thus it was not skewing the data unnecessarily in one direction and was therefore not the cause of the significant negative correlation found (recall: rs=-0.341, df=36, p=0.036, recognition: rs=-0.175, df=36, p=0.293).\n\nThe ages of the participants were not greatly varied, with thirty-six out of thirty-nine participants aged nineteen to twenty-one, one participant aged twenty-three, one participant aged thirty and one participant not stating their age. The removal of the data for the participant aged thirty did not change the statistical significance of the original result, thus the greater age of this participant in comparison to the others was also not the cause of the significant negative correlation found (recall: rs=-0.387, df=36, p=0.016, recognition: rs=-0.251, df=36, p=0.129). Furthermore, the imbalance in the number of male and female participants (seven male, thirty-two female) is unlikely to have caused any bias in the data as there was no significant difference between the two genders in the mean values for the recall errors (Wilcoxon rank sum test: W=114, p=0.956), recognition errors (Wilcoxon rank sum test: W=97.5, p=0.605) nor the categorisation test scores (Wilcoxon rank sum test: W=102, p=0.727).\n\n\nDiscussion\n\nOur findings show a trade-off between word categorisation ability and semantic false memory susceptibility, so that individuals that make more errors on the false memory test make fewer errors on the categorisation test, and vice versa. Thus our results cannot simply be explained by differences in level of education, literacy, vocabulary or intelligence. If such an underlying factor would have explained performance on both tasks, then superior performance on one task would have been a predictor of superior performance on the other task. For example, short term memorisation of word lists recruits working memory, which is often regarded as a general predictor of intelligence (Oberauer et al., 2005; Oberauer et al., 2008) and likewise the categorisation tests used here are typical components of standardised intelligence tests (Wechsler, 2004; Wechsler, 2008). Thus one might have predicted a positive correlation of error scores in both tasks if an underlying single factor such as intelligence would explain the data. However, the correlation of error scores in the two measured tasks was negative. Thus even though this study is clearly correlative in nature, and therefore does not allow us to conclude with certainty that the two performances are based on the same underlying mechanisms, it is intriguing that having a lower tendency to generate false memories comes at a cost, i.e. lower categorisation scores.\n\nTo date the majority of scholars interested in false memories have focused on factors which may exacerbate or reduce the occurrence of such memory errors (Dodson et al., 2000). The adaptive nature of the human memory system as a potential reason for the occurrence false memories has been suggested (Schacter, 1999; Schacter, 2001), yet the ultimate reasons for their existence has been infrequently explored empirically. More recently, however, evidence has grown for links between individuals’ differing susceptibilities to false memories and their variations in a range of cognitive features. False recall and/or recognition rates in a DRM paradigm have been shown to vary with individuals’ variations in levels of vivid mental imagery (Winograd et al., 1998), specific area expertise (Baird, 2003; Castel et al., 2007), working memory capacity (Watson et al., 2005) and need for cognition (the degree to which an individual actively engages in cognitive tasks) (Graham, 2007).\n\nAdditionally it has been shown that when survival-related (i.e. evolutionarily relevant) information is used in a list-learning paradigm, increased susceptibility to false memories occurs. Howe & Derbish (2010) found that when participants are asked to process words for their survival value and when the words presented were themselves survival relevant (i.e., ‘death: burial, casket, cemetery, funeral, grave, life, murder, suicide, tragedy, widow), veridical and false recognition were significantly higher (leading to an overall decrease in net accuracy) than when the words viewed were neutral or negative and were processed for pleasantness. They concluded that whilst it does not at first seem adaptive for survival-related memories to be less accurate and in fact be more prone to false intrusions than other types of memory, it does make sense if considered as a by-product of the adaptive processing of information related to survival. Howe & Derbish, (2010) argue that during the processing of information related to survival, any related information in memory is then primed, which may or may not be false, but that this information is then used to guide attention to other survival-related items, which may be crucial in the current situation (Howe & Derbish, 2010).\n\nIt has even been postulated that this greater inaccuracy may actually have adaptive significance, being more helpful in real-world scenarios. For example, in responses to predation threat, false alarms, such as generalising to a large set of cues that might indicate predator presence are clearly less detrimental errors than missing predator presence based on interpreting predators’ cues too narrowly (Howe & Derbish, 2010). Thus our finding of a significant positive correlation between susceptibility to semantic false memories in a free recall DRM paradigm and word-based categorisation ability, with the creation of these errors a by-product of our ability to group words, is in keeping with recent explorations of the adaptive conditions related to the phenomenon of false memories.\n\nWhilst the age range of the subjects tested was narrow (nineteen to twenty-one years old in the majority) many of the key studies using the DRM paradigm have used only participants also of average undergraduate college study age (Roediger & McDermott, 1995; Stadler et al., 1999). Additionally the only significant difference in spontaneous false memory creation, caused by the DRM paradigm that is known to occur between participants of different ages, is between children and adults. Several studies have shown that children are less prone to these memory errors, with an increase in their propensity occurring during both childhood and early adolescence (Brainerd et al., 2002; Brainerd et al., 2004; Forrest, 2002). As such, inferences made from our findings are not just applicable to young adults but should also be pertinent to the ‘average’ adult population as a whole.\n\nOur result of a significant negative correlation between individuals’ errors on a categorisation test and their susceptibilities to semantic type false memories during free recall demonstrates that false memories, to some extent, might be a by-product of our ability to learn rules, categories and concepts. For example, once we have learnt the concept/category of mammals, we can identify new animals as members of this category even if we have never seen them before. In this case, labelling the new animal as mammal is not based on false classification, but a correct one based on category membership: the simple flipside of the DRM paradigm, where inferences based on concepts and categories are classed as errors. Thus, our findings add to the increasing body of literature that proposes that false memories might be an inevitable by-product of adaptive cognitive processes as is the case with other memory aberrations (Abbott & Sherratt, 2011; Beck & Forstmeier, 2007).\n\n\nData availability\n\nF1000Research: Dataset 1. False memory susceptibility and categorisation ability, http://dx.doi.org/10.5256/f1000research.4645.d31516 (Hunt & Chittka, 2014).", "appendix": "Author contributions\n\n\n\nLC conceived the study. KH designed the experiments and carried out the research. Both authors were involved in writing all versions of the manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was funded as part of a PhD studentship provided by the Natural Environment Research Council (NE/H525089/1).\n\n\nAcknowledgments\n\nThe authors acknowledge S. Ali and R. Begum for their help with resource construction and data collection.\n\n\nReferences\n\nAbbott KR, Sherratt TN: The evolution of superstition through optimal use of incomplete information. Anim Behav. 2011; 82(1): 85–92. Publisher Full Text\n\nAvarguès-Weber A, Deisig N, Giurfa M: Visual cognition in social insects. Annu Rev Entomol. 2011; 56: 423–443. PubMed Abstract \| Publisher Full Text\n\nBaird RR: Experts sometimes show more false recall than novices: A cost of knowing too much. Learn Individ Differ. 2003; 13(4): 349–355. 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Publisher Full Text" }	[ { "id": "5492", "date": "08 Aug 2014", "name": "Rob Nash", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting article that has good relevance to ongoing debates on the adaptiveness of memory errors. The methods and analyses are appropriate and the authors have generally represented the scientific literature well. I have a few minor suggestion for improvement, as follows: In the paragraph beginning “While such tests might be viewed as rather remote from real-life situations..” it should be noted that there are several more studies in which errors in different false memory paradigms are correlated either weakly (e.g., Zhu et al., 2013) or not at all (e.g. Ost et al., 2013). The line “Clearly false memories cannot in themselves be useful” is disputable – several studies now show positive consequences of distorted memories, see e.g. Howe, Garner & Patel, 2013; Bernstein & Loftus, 2009). Line “It is therefore possible that memory errors are an inevitable fluke of a powerful, adaptive cognitive phenomenon” – I would prefer to say “some memory errors” – there is a broad literature on other adaptive reasons why memory errors occur, see e.g. Newman & Lindsay (2009). Same point applies to the very final sentence of the Discussion. Why wasn't the order of the two tasks counterbalanced? Is it plausible that the first (memory) task might have primed a particular mindset in participants that affected their categorization performance? I’d suggest the addition of a little discussion of this point. Data analysis – which data departed from normality? The authors have reported a normality test but it isn't clear to which variable this test pertains. The line “A significant negative correlation was still found for free recall and a moderate negative correlation still found for recognition” – the authors should reiterate explicitly that the latter correlation was non-significant.", "responses": [ { "c_id": "1038", "date": "17 Oct 2014", "name": "Lars Chittka", "role": "Author Response", "response": "We thank the referee for the constructive comments. We have taken all of them on board in the revised version. We have also added the five additional references indicated by the referee, and modified the text in line with the contents of these publications. We have added the requested information about non-normality of some of the data.The referee asks why we have not used a counterbalanced design, so that in addition to setting the false memory test first and the categorisation test second, we might have reversed the order. This would indeed be a useful procedure to exclude the possibility that any correlation could be caused by priming, occurring as a result of words occurring in both tests. However, to address the potential complication of priming, we had already reanalysed the data excluding those categorisation test questions that contained words previously presented in the DRM word lists, and non-presented as one of the critical non-presented words. However, even after removal of all individual tasks where such words occurred, the overall results remained unaltered (see results section)." } ] }, { "id": "5357", "date": "16 Sep 2014", "name": "Tom Smulders", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper, Hunt and Chittka show that people who are better at a categorization task also tend to produce more \"memory errors\" on the DRM paradigm, in which 15 words are shown which are strongly associated with a non-presented word (which is then often (wrongly) recalled or recognized later by the participants). The experiment has been conducted properly (the issue of counterbalancing has been addressed by the priming re-analysis) and the results are clear, although the effect size does not seem large.The conclusions the authors draw from this result seem eminently reasonable, although they may go a bit beyond the immediate data. After all, they specifically picked a form of false-memory testing that relies heavily on the fact that words in a semantic category are tightly associated with each other. It is then not a great surprise that people who are good at keeping words from the same semantic categories together also show more memory errors. It might have been interesting to ask people to categorize on different criteria than semantic (e.g. based on the letters they contain). Even though this is still a test of finding odd words out in a group, it does not call on semantic categories...The introduction is probably setting up a bit of a straw man. I think false memories as discussed here should be limited to those in episodic memory. Even the DRM is an episodic memory task (recall the unique list you've just been presented with), which is influenced by semantic associations between words. Therefore, any discussion of selective pressures on memory accuracy should be based on episodic memory alone. And there are lots of debates about what episodic memory is for, and indeed whether accuracy is the most important part of episodic memories. False memories in episodic memories often come about by \"intrusion\" of more common events into a unique episode. If the common events are that common, the unique exception may not be important to remember and indeed may interfere with the (adaptive) application of a learned rule. So there may be many arguments against the idea that memory should always be accurate. Nevertheless, this does not take away from the data or the final conclusions of this paper.", "responses": [ { "c_id": "1039", "date": "17 Oct 2014", "name": "Lars Chittka", "role": "Author Response", "response": "We thank the referee for the constructive comments. We agree with the referee that one function of cognition (probably not just episodic memory) is to extract commonalities between distinct individual memories, learn rules, and to categorise objects and events by common properties, and to classify novel objects or events into existing frameworks. Certainly such rule-learning and classification abilities are adaptive, and we have thus deleted the statement that “false memories cannot in themselves be useful”.Nonetheless, it is probably fair to say that many of the contexts in which false memories occur ‘naturally’, as well as the common experimental paradigms (not just ours), rely on rule learning and classification abilities. Witnesses of a nocturnal street robbery might describe the perpetrator as a hooded teenager, when it later turns out that the assailant was middle-aged and balding. While such false memories create enormous difficulties for the police and criminal courts, the witnesses may have made of use of their (typically, but clearly not always, adaptive) ability to learn rules about settings or conditions that, at least potentially, indicate danger. From the earliest explorations of false memories, such as the transmission chain studies in Bartlett’s work, it was investigated how distorted memories fall in with previously memorised rules, cultural norms etc. Whether this means that individuals that are better at extracting common properties incur a cost when it comes to storing large number of individual items/memories is precisely the subject of this study. We agree with the referee that it might sometimes be more useful to memorise common events than individual ones, or indeed to extract the commonalities between events and discard their idiosyncrasies, and this is hopefully clearer in the revised version." } ] } ]	1	https://f1000research.com/articles/3-154
https://f1000research.com/articles/2-66/v1	28 Feb 13	{ "type": "Research Article", "title": "Trading green backs for green crabs: evaluating the commercial shellfish harvest at risk to European green crab invasion", "authors": [ "Megan E Mach", "Kai MA Chan", "Kai MA Chan" ], "abstract": "Nonnative species pose a threat to native biodiversity and can have immense impacts on biological communities, altering the function of ecosystems. How much value is at risk from high-impact invasive species, and which parameters determine variation in that value, constitutes critical knowledge for directing both management and research, but it is rarely available. We evaluated the value of the commercial shellfish harvest that is at risk in nearshore ecosystems of Puget Sound, Washington State, USA, from the invasive European green crab, Carcinus maenas. We assessed this value using a simple static ecological model combined with an economic model using data from Puget Sound’s shellfish harvest and revenue and the relationship between C. maenas abundance and the consumption rate of shellfish. The model incorporates a range in C. maenas diet preference, calories consumed per year, and crab densities. C. maenas is likely to prey on commercially harvested hardshell clams, oysters, and mussels, which would likely reduce additional revenue from processing and distribution, and the number of jobs associated with these fisheries.The model results suggest possible revenue losses of these shellfish ranging from $1.03-23.8 million USD (2.8-64% losses), with harvesting and processing losses up to $44 million USD (40%) and 303 job positions each year associated with a range of plausible parameter values. The broad range of values reflects the uncertainty in key factors underlying impacts, factors that are highly variable across invaded regions and so not knowable a priori. However, future research evaluating species invasions can reduce the uncertainty of impacts by characterizing several key parameters: density of individuals, number of arrivals, predation and competition interactions, and economic impacts. This study therefore provides direction for research to inform more accurate estimates of value-at-risk, and suggests substantial motivation for strong measures to prevent, monitor, and manage the possible invasion of C. maenas.", "keywords": [ "green crab", "shellfish harvest", "nonnative species", "invasive species" ], "content": "Introduction\n\nIn coastal ecosystems, preventing and mitigating the spread and impacts of nonnative species has become a global priority1,2. While many nonnative species have little to no measurable impact on their invaded regions, a few have caused great economic and ecosystem harm3,4. The impacts from these few invasive species can affect ecosystem function and thereby reduce the benefits that ecosystems provide for people5–9. In the USA, the cost of invasive species impacts has been estimated at over $120 billion USD per year3,10. For example, zebra mussel, Dreissena polymorpha, invasions in the Laurentian Great Lakes have fouled industrial water intake pipes, enhanced macrophyte growth and reduced water turbidity, altering habitat conditions for native fish–all of which have resulted in economic losses to local communities11. With limited funds to manage and research coastal ecosystems, calculating the value-at-risk (the losses that might accompany the establishment of a high-impact invasive species prior to introduction) for areas not yet invaded may justify the allocation of resources to prevent, mitigate, or further understand invasive impacts12,13.\n\nThe estimation of value-at-risk from invasive species should be distinguished clearly from a purpose of prediction. Rather than a statement of what is expected to occur, value-at-risk can provide decision-makers with a sense of what might plausibly be lost to invasion without prevention or mitigation. Whereas in finance, value-at-risk is often a monetary quantity subject to loss with a given probability14, the concept might also be useful in settings where data limitations restrict the explicit assignment of probabilities. For example, a decision-maker faced with the decision whether to fund action to prevent biological invasion, or to put in place mechanisms to mitigate losses should invasion occur, might only wish to know what value–in revenues, jobs, etc.–might plausibly be subject to loss, based on our current understanding and its limitations. For such decisions, which are faced every day, waiting for better data or a more sophisticated model may not be an option. Just as this quantity of value-at-risk can motivate management action, it can also motivate research to improve estimates of value-at-risk, and perhaps even enable prediction.\n\nEconomic value-at-risk can be seen as a product of two components: ecological (plausible ecological changes that might result from the introduction of a known invader) and economic (the economic costs that might be associated with the above ecological changes or resulting mitigation). Ecological consequences have been assessed mainly as projected post-invasion impacts or pre-invasion estimated ecosystem changes15–17. For example, ecological impact estimates of the European green crab, Carcinus maenas, suggest a future loss of valuable habitats and native species abundance in invaded areas, while larvae of the green crab may also provide food resources to migrating salmon in the northeast Pacific15.\n\nThe economic consequences of invasive species can be estimated through damages to resources3,4,18. However, many studies that evaluate economic costs do so with considerable simplification of important ecological processes, potentially misrepresenting costs19–21. For example, damages have been measured as reduced fishery values since time of invasion22,23, but these estimates do not consider other potential causal factors or variation in key traits of the native or invading species, such as varying invasion densities, predation rates or varying spatial distribution of native species. Economic valuations can better represent impacts of nonnative species if they incorporate specific information on both the native and invasive populations and their potential interactions20.\n\nThere are great uncertainties in predicting impacts of species invasions24,25, in part because impacts vary across space and time and are otherwise context-dependent26–30. To be useful, evaluations made before invasions occur should incorporate economic costs and uncertainties associated with the invasion’s possible ecological consequences21,31. If value-at-risk estimates are structured to enable explicit assessment of the uncertainties associated with key parameters, even a coarse understanding of potential impacts can yield useful assessments. Ecological-economic models offer the opportunity to estimate economic changes that include these uncertainties19. Though the value-at-risk of predicted invasions has not frequently been calculated, preemptive management strategies that incorporate ecological modeling could have considerable economic benefits13,19. For example, if early preventative efforts had been funded to prevent invasion of the rusty crayfish, Orconectes rusticus, Vilas County, Wisconsin, USA, it would have protected $6 million USD in fisheries harvest revenue during the 30 years since invasion13. These calculations serve a critically important purpose of aligning future research and management25.\n\nHere we assessed the value-at-risk for commercial shellfish harvest in nearshore ecosystems of Puget Sound, Washington State, USA, from the green crab, C. maenas. A species already regarded as a threat to ecosystems in Puget Sound32,33. To assess value-at-risk, we combined an ecological model of risk to shellfish harvest biomass by C. maenas with an economic model demonstrating risk to current shellfish harvest revenue. The secondary economic effects were predicted using benefit transfer from a similar neighbouring region, British Columbia, Canada (BC). Furthermore, we characterized the extent to which variation in key parameters influenced the resulting value-at-risk.\n\n\nMethods\n\nNonnative species with broad physiological tolerances and diverse diets, such as Carcinus maenas, are well suited to take advantage of available resources and out-compete native species34. C. maenas is a generalist predator that, in one study, was found to consume species from at least 104 families and 158 genera within 14 animal and 5 plant and protozoan phyla35, though it most commonly feeds on bivalves36,37. In feeding trials, C. maenas was able to feed more generally and consume a greater biomass than several native northeast Pacific Cancer crab species35. Green crab have destroyed artificial shellfish beds and consumed juvenile bivalves and Cancer crabs throughout northern New England and the Canadian Maritimes (review in23), reducing profits from the northwest Atlantic shellfish industry by as much as $22.6 million USD each year since its introduction38. Studies from previous invasions of C. maenas suggest Puget Sound’s commercially harvested bivalve species are a likely target for green crab predation23,38,39. Green crab invasion in Puget Sound is predicted to impact economically important hardshell clam, oyster and mussel species36 all of which bring in millions of dollars in direct and indirect revenue to the Puget Sound region40,41.\n\nIn addition to the risk to shellfish, green crab risk assessments from Puget Sound and a field experiment in the northeast Atlantic have demonstrated the negative effects of green crabs on natural habitats; for example, Zostera marina eelgrass habitat is destroyed and associated food webs are disrupted by green crab invasion15,42. The loss of these habitats could have secondary impacts on many of the benefits that eelgrass provides to near-shore estuaries, such as an essential spawning habitat for herring, and nursery grounds for many commercially important fish and shellfish43,44. Clearly, not all impacts of green crab are likely to be negative: Coho salmon (Oncorhynchus kisutch) were predicted to benefit from invasion by feeding on the larvae of green crab, although this projection is associated with great uncertainty15 and is not included in the current model.\n\nNative to northern Europe, C. maenas has established populations in North America45, South Africa46, Japan47, Argentina48 and Australia49. In North America, the green crab was first found on the northern Atlantic coast in 1899, and it has since expanded to cover 1000 kilometers of coast from Virginia in the south to a still-expanding range on Prince Edward Island45,50. In 1989, C. maenas is believed to have reached San Francisco Bay in the northeast Pacific via ballast water from populations on the northwest Atlantic coast51.\n\nSecondary spread of green crab north along the northeast Pacific coastline has been correlated with increased seawater temperatures and north-running coastal currents during the 1998 El Nino event, making it particularly likely that future climate change will allow the crab to invade new areas of coastline52,53. The green crab is limited by temperature and salinity, surviving in water temperatures ranging from 0°C to 30°C and salinities of 4 to 34 mV/V, although reproduction and larval survival occur in a more limited range than the adults (review in35). Predictions based on these physiological limitations suggest that under current conditions green crab will continue expanding northward from its current northern extent of Vancouver Island until it reaches the Aleutian Islands45, and that it may enter the contiguous waters of Puget Sound and the Strait of Georgia, BC, either by secondary introduction events from ballast release from large shipping freighters or through natural larval dispersal during one of the next El Nino events (Figure 1)54. Puget Sound is a large coastal estuary where extensive mudflats, eelgrass beds and warmer inland waters could provide optimal habitats for C. maenas foraging and reproduction.\n\nThe current nonnative distribution along the coast is indicated by a broad, stippled polygon, while the potential distribution of the species is plotted in black. Figure and Maxtent potential distribution model from deRivera et al.83, figure altered to clarify the absence of C. maenas from Puget Sound. This figure has been adapted and reproduced with kind permission from Diversity and Distributions, © 2011.\n\nTo estimate the harvest for Puget Sound’s commercial shellfish industry, we obtained commercial harvest data for 2009 from PacFIN (http://pacfin.psmfc.org/pacfin_pub/data.php) on May 26, 201055. These data included species information for all clams, mussels and oysters commercially caught and farmed in Puget Sound, harvest biomass (kgs) and total revenue (USD $) for 2009 (summarized in Table 1). Data were apportioned to individual Puget Sound Partnership (PSP) action areas56 (Figure 2) by intersecting these with Washington Department of Fish and Wildlife (WDFW) and Department of Health 2010 approved commercial shellfish growing areas (http://ww4.doh.wa.gov/gis/gisdata.htm)57 using the ArcGIS 9 Intersect tool (Figure 2; completed by Mark Plummer, NOAA). Data were assigned to PSP action areas assuming the harvest occurs uniformly throughout the growing area. Commercial data included harvest (kilograms year-1) and total revenue (USD year-1) for hardshell clams (Venerupis philipinarum and Protothaca staminea), oysters (Crassostrea virginica and C. gigas) and mussels (Mytilus spp.) in six PSP action areas (hereafter, harvest areas). Shellfish biomass within each species group was summed to create a total estimated biomass for each harvest area. This biomass was then used to calculate an average cost per kilogram of shellfish (USD kg-1). These data were used as the baseline estimate for current shellfish harvest and revenue before green crab invasion.\n\nBoundaries of the Puget Sound Action Areas, each of which represent a unique watershed and harvest region as designated by the Puget Sound Partnership56. Washington State Department of Health's approved commercial shellfish growing areas for 201057 are highlighted in green. Included in this study are data on shellfish harvest biomass and revenue from all Action Areas except South Central Puget Sound.\n\nThe seven harvest areas of Puget Sound with complete commercial harvest data for each taxa group were Hood Canal, North Central Puget Sound, Whatcom/San Juan, Strait of Juan de Fuca, Whidbey Island, South Central Puget Sound, and South Puget Sound. South Central Puget Sound only represents 0.4% of the total harvest area and was not included in the analyses.\n\nShellfish species in Puget Sound are commercially harvested from mudflats or grown in aquaculture farms. All species evaluated in this study spend a portion of their life-cycle in the near-shore where they are susceptible to green crab predation:\n\na) Hardshell clams–Manila (V. philipinarum) and native Littleneck (P. staminea)–are harvested on tidal flats throughout Puget Sound at sediment depths of less than 15 cm. Hardshell clams are either raked off beaches where they grow naturally or their beds are “seeded” (seed clams are sown onto beaches leased from Washington State). These two species make up 98% of total hardshell clam harvest.\n\nb) Oysters–European (C. virginica) and Pacific oyster (C. gigas)–these species are harvested from populations that grow without assistance in the high subtidal/low intertidal and on aquaculture farms where they are grown directly on mudflats, on racks sitting on the bottom substrate, or suspended under floating rafts.\n\nc) Blue mussels (Mytilus spp.) grow on rocks in the high subtidal/low intertidal and are harvested on state approved beaches and on aquaculture farms grown on racks sitting on the bottom substrate or suspended under floating rafts.\n\nTo model the impact of C. maenas predation on shellfish in Puget Sound we applied the following simple linear model to estimate the total kilograms of each of three species of shellfish species groups (hardshell clams, oysters, mussels) consumed each year by C. maenas (Consumption):\n\nConsumption = (Area x Den x Cal x Diet)/Cal kg-1\n\nThe variables Area, Den, Cal, Diet, and Cal kg-1 are defined below.\n\nArea: Commercial shellfish harvest area (km2) in Puget Sound (Area) was estimated as 523.71 km2 by tracing polygons around the shellfish harvest areas in the Washington State Department of Health Annual Inventory Growing Areas Map58 within each of the five PSP action areas (Figure 2) using ImageJ (Table 1)59.\n\nDen: Density of adult C. maenas invading the harvest areas (crabs km-2) was represented as a range of possible invasion densities. The high density estimate, 100,000 km-2 (High), represents maximum adult green crab densities from one study in C. maenas’s native range, in Sweden60. The high estimate for population density averaged over time is justifiable given that densities are sometimes considerably higher in invaded ranges due to lower predation pressures and greater available resources61,62. The low density estimate, one order of magnitude less than high at 10,000 km-2 (Low), is equivalent to average adult green crab densities in their native range60. We selected a medium density estimate of 50,000 km-2 (Medium), roughly midway between high and low estimates. We refer to studies of densities from the native range because studies from invaded regions use catch per unit effort (CPUE) to estimate invasion densities34,48,63,64. CPUE is difficult to translate to a density of individuals per area because studies use different traps or dredges, set them using different methods, and leave them to catch crab for differing lengths of time.\n\nCal: The number of calories consumed by each adult C. maenas per year was estimated by extrapolation from laboratory diet studies. The number of calories mg-1 (ash-free dry weight, AFDW) of shellfish meat from an individual adult shellfish (clams, 6.15 cal mg-1; oysters, 4.85 cal mg-1; mussels 5.47 cal mg-1; as reviewed by65) was multiplied by the number of mg per day a C. maenas (25 to 32 cm carapace) was found to consume. However, green crab prefer to prey on juvenile shellfish66,67 while shellfish harvest biomass is of adult shellfish, estimates based on harvested adult may overestimate predation impacts. In addition, this model assumes all harvested shellfish biomass is accessible to green crab predation. In practice, aquaculture using racks suspended above the bottom sediment to grow shellfish will limit or prevent predation, and clams may burrow deeper than green crab can dig through the sediment thus reducing predation on these species; on the other hand it is possible that deep-burrowing clams may be economically infeasible to harvest.\n\nDiet: The proportion of the C. maenas diet consisting of each shellfish species group being modeled was estimated as ranging from 0.20 to 0.35. Grosholz and Ruiz37 demonstrated that green crab diet is similarly dominated by bivalves in each region it has invaded. Our estimate range for diet assumes that 60–100% of C. maenas’s diet will be harvestable hardshell clams, oysters, and mussels, thus allowing for other species to comprise up to 40% of C. maenas’s diet37,68. Estimates of Diet do not include prey-switching by green crab, which may occur as the preferred shellfish biomass is reduced during invasion.\n\nCal kg-1: We used calories mg-1 AFDW (ash free dry weight) of shellfish meat (as in Cal; reviewed by65), to calculate the number of calories per kilogram of shellfish (Cal kg-1). AFDW was converted to wet weight (WW), the unit of biomass for shellfish harvested in Puget Sound, using conversions reviewed in Ricciardi & Bourget69. Conversion estimates were different for each species group and include a 95% confidence interval for all estimates that were made using more than one study. We used AFDW/WW conversion estimates of M. edulis for mussels (2.5–6.7), C. virginica for oysters (1.7), and the average conversion estimates of all bivalves for hardshell clams (5.2–6.4; combined for the two hardshell clam species).\n\nWe accounted for uncertainty in these parameters in two ways. First, where the range in potential data for the variable is large, as described below for crab density (Den) and calorie diet for each crab (Cal), we set three broad estimates (high, medium, and low) across the range of data from the literature and modeled these as separate scenarios for C. maenas consumption. Second, where the range in data uncertainty was smaller, we represented the uncertainty through randomization within scenarios defined as above, by choosing values across a uniform distribution for harvest area (Area), the proportion of each crab’s diet that is the shellfish being analyzed (Diet), and the number of calories per kilogram of each shellfish species (Cal kg-1 ). Den and Cal parameters are arguably not independent: certain values of one have the potential to constrain values of the other. This is an important possibility for which there is insufficient data to model the relationship given the presence of other complicating factors, such as limited recruitment of harvestable shellfish (see Discussion).\n\nTo implement the randomization for Area, Diet, and Cal kg-1, we used a Markov Chain Monte Carlo (MCMC) algorithm using R software70. Data were generated by resampling 10,000 times within the constraints described below for each parameter in the consumption model. To implement the continuous variation between the upper and lower bounds for Area and Diet parameters, we used the runif function in R. Runif samples randomly from a uniform distribution between the upper and lower bound parameters. For Area we assumed this range to be relatively small around the single estimate above, ± 75 km2, as some error was possible as a result of apportioning of WDFW Shellfish Management and Aquaculture areas to PSP action areas and the measurement of harvest areas using ImageJ. The Diet proportion was evenly sampled between 0.20 and 0.35 for each shellfish species. Considering C. maenas has not yet invaded Puget Sound nor had its predation preference tested for any of these shellfish species, precise estimates of its diet preference with the species it will encounter in the Puget Sound region were not available. In addition, these values will likely vary depending on the availability of shellfish and ease of predation. We attempted to partly reflect this by allowing diet preferences values to vary evenly across a range of proportions. We calculated the range in number of calories per kilogram of each shellfish species using the range of conversion estimates between AFDW and WW for each shellfish species group (Cal kg-1). An example of the R code used for these analyses is available in the Supplementary Materials.\n\nWe estimated the impact of green crab on harvested shellfish for high, medium, and low invasion densities at high, medium, and low calorie diets by calculating the error for mean consumption at each density (95% confidence interval from randomizations within each scenario). This calculation resulted in an upper and lower estimate of consumption of total shellfish biomass in harvest areas for each combination of invasion density and calorie intake. Annual harvest of each shellfish species was then estimated as the total baseline annual harvest minus the upper and lower consumption estimates. These methods were repeated for each of the three shellfish species groups: hardshell clams, oysters and mussels.\n\nIn order to represent parameter combinations that fall between the consumption scenarios for Den and Cal, we performed a partial sensitivity analysis of parameters in the model71. In this analysis, we allowed Den and Cal to range freely from zero to high estimates, with a uniform distribution, and held the other parameters to the same constraints as described above, except Diet of harvested shellfish was estimated as 60% to 100% of green crab diet to include all three harvested shellfish species groups. Data were again sampled with the MCMC algorithm using R software70, with 10,000 replicate samples within the parameter constraints.\n\nWe considered the primary economic value of shellfish harvest in terms of existing harvest revenue (landed value) and secondary economic value in terms of processing and distribution value and direct impacts from primary and secondary value in terms of labour income and employment. To evaluate the primary economic value-at-risk from green crab predation on shellfisheries in Puget Sound, we estimated the loss of existing harvested revenue from the total revenue of hardshell clams, oysters and mussels for high, medium, and low densities of green crab and across high, medium, and low calorie diets. Loss of shellfish harvest revenue, which is calculated as USD kg-1, was assumed to decrease in parallel with the loss of harvested shellfish biomass to green crab predation as estimated by the consumption model.\n\nFurther estimates of the secondary economic value from direct impacts of Puget Sound shellfisheries: processing, distribution, and labour values, were made using benefit transfer methods as data on secondary value were not available for shellfish species in Puget Sound. Thus, we compared the known revenue of Puget Sound’s shellfish harvest to an analysis done on the value of shellfish harvest in BC, an adjacent region to Puget Sound (Table 2)72. GSGislason & Associates Ltd72. estimated the value of all of BC’s shellfisheries (in CAD) in 2005 for harvesting of shellfish, which involves the use of beach harvest, diving and other gear and aquaculture of shellfish from seed to market size. Secondary economic values were estimated for all fisheries species (including both fish and shellfish). To calculate direct impacts of shellfisheries alone, we assumed the ratio of the secondary value as compared to harvesting value of shellfisheries was the same as the ratio of the secondary value as compared to the fish and shellfisheries harvesting value.\n\nSecondary values were derived from primary values (2009 Puget Sound shellfish harvest value in millions USD, for capture and aquaculture) and % shellfish losses (in parentheses, as output from the ecological model), using benefit transfer from British Columbia in 200572.\n\nSecondary values were calculated for 1) the processing margin, which includes transportation from sea to processing plants and the processing of raw shellfish, 2) the distribution margin, defined as the delivery of these processed shellfish products to consumers through wholesale and retail food channels, 3) the direct impacts of the seafood industry on labour income, which includes wages, salaries, and employer contributions to health and dental plans, pension plans, etc., and 4) employment years in persons per year (PY)73. To estimate the secondary value of shellfisheries in Puget Sound, we then assumed the ratio of secondary economic value to shellfish harvest revenue is the same for shellfisheries in Puget Sound as in BC (Table 2). This ratio was also assumed to be the same for commercial shellfish processing, distribution, labour income and employment in Puget Sound. We then estimated C. maenas impact on these secondary economic values at high, medium, and low invasion densities and high, medium, and low calorie diets. We assumed the loss of shellfish harvest revenue and secondary values decreased at the same rate as more green crab invade and consume increasing calories per crab as estimated by the consumption model.\n\n\nResults\n\nThe baseline for total shellfish harvest in Puget Sound for 2009 for hardshell clams, oysters and mussels recorded by PacFIN55 was 6.05 million kgs of shellfish, with a landed harvest value of $37.26 million USD (Table 1). Hardshell clams had the highest biomass harvested out of the three species groups (3.4 million kgs) and the greatest associated revenue, $18.3 million USD, even though oyster species are valued per kilogram at almost twice that of hardshell clams, $10.44 kg-1 compared to $5.38 kg-1 respectively (price does not include the shell).\n\nUsing the consumption model, we estimated harvested shellfish biomass and total shellfish harvest revenue associated with scenarios of low, medium and high calorie diets and densities for green crab: the medium-medium (Cal-Den) scenario yielded a value-at-risk estimate of 0.54 million kgs and $3.72 million USD in harvest revenue, a loss of 9.0%; the low-low scenario suggested a minor loss of only 0.04 million kgs and $0.08 million USD, a loss of 0.3%; and medium-high, high-medium, and high-high scenarios suggesting losses of at least 0.99 million kgs and $6.76 million USD (Table 3 and Figure 3). Mussels were the only shellfish to reach an estimated value loss of 100% for the high-high scenario (Figure 3c). Note that these losses pertain to losses of harvested biomass, not of all prey organisms, allowing for the possibility that green crab might eliminate harvestable biomass without causing local extirpation (see Discussion).\n\nThe baseline shellfish biomass harvest (millions) and direct harvest revenue value (millions, 2009 USD) are compared to shellfish harvest under three scenarios of green crab densities (low, medium, and high densities) and of calories consumed by green crab each year (low, medium, and high calorie diets). Percent losses (% loss) represents change in total shellfish biomass from the baseline.\n\nMean harvested shellfish biomass (kgs) and revenue (USD) at increasing levels of green crab densities for three shellfish species groups: a) hardshell clams, b) oysters, c) mussels at three increasing levels (dotted light grey, low; dark grey dashes, medium; black solid line, high) of calories consumed by each green crab year-1.\n\nThe Monte Carlo sensitivity analysis demonstrated the relationship between density and calorie consumption rate on projected loss of shellfish harvest biomass (Figure 4). If crabs invade at high densities but consume calories at a low rate, or vice versa, they would likely have a limited effect on shellfish harvest. However, at high densities and high calorie intake rates the slope of the shellfish harvest loss is steep and green crab will likely greatly reduce shellfish harvest biomass.\n\nLoss of shellfish harvest biomass (kgs year-1) as a function of calories consumed each year and the density of C. maenas km-2 in harvest areas. Assumes 60% to 100% of green crab diet is made up of hardshell clams, oysters and mussels.\n\nThe harvest revenue associated with the total biomass for these three species of shellfish in Puget Sound is $37.26 million USD (26.6% of BC’s revenue). Estimated loss at low, medium and high green crab calorie diets was up to 3%, 47% and 64% at the highest green crab density of 100,000 green crab per km2. Specific values for change in known harvesting values and estimated processing margin and distribution margin values, as well as potential change in labour income, are presented in Table 2. Employment associated with shellfish harvest and farming, processing, and distribution is estimated around 687 person-years per year (PYs). Green crab invasion at highest densities is estimated to reduce these PYs by 12 at the lowest calorie diet, and by 303 at the high calorie diet (Table 2).\n\n\nDiscussion\n\nBy assessing the value-at-risk for commercial shellfish harvest in nearshore ecosystems of Puget Sound, Washington from the green crab C. maenas, we estimated a range of possible losses that included, at highest invasion densities for three scenarios of calorie-intensity of diets, up to 0.15–4.46 million kgs of shellfish worth $1.03 – $23.8 million USD, a loss of 2.2–56%. For highest calorie diets, across a range of invasion densities shellfish harvest loss estimates ranged from 0.45–4.46 million kgs worth $2.5–23.8 million (6.8–56%). This corresponds to a total loss of revenue, including processing and distribution margins, of $5.1–44 million USD (7–40% loss) and a loss of 47–303 jobs (PYs). Estimated value-at-risk is likely to vary across Puget Sound harvest areas due to refuges for shellfish populations and uneven distributions of green crab due to habitat variability. Green crab appears to have the potential to reduce shellfish biomass enough to effectively halt the shellfish harvest industry completely at the highest invasion densities or highest calorie diet rate, since low shellfish population densities result in shellfish harvest area closures.\n\nIf green crab significantly contributes to reducing biomass, hardshell clam populations may be reduced below allowable harvest biomass. Thus, reducing populations of harvested shellfish to zero within the consumption model does not suggest there are no shellfish remaining, but rather that when shellfish populations are sufficiently reduced there is no more commercial harvest of shellfish. In addition, green crab are opportunistic feeders that are likely to feed on other species when preferred bivalve abundance becomes low. So although green crab may decrease shellfish population densities, they are likely to feed on whatever species are the easiest to access, maintaining bivalve populations at low densities but not extirpating the populations64. The reduction of shellfish biomass also has the potential to limit recruitment of shellfish by reducing the population of reproductive adult shellfish and by preying on newly settled juvenile shellfish. These negative feedbacks may result in greater losses to shellfish then we have estimated.\n\nAlthough the range in values is broad it appropriately represents the uncertainty in key factors associated with green crab invasion impacts, factors that are highly variable across regions. As such, the most extreme estimates of shellfish harvest loss cannot be wholly excluded from consideration: these estimates are based on current understanding of green crab populations in other regions, with several caveats. Lower calorie estimates are likely more similar to impacts from previous invasions than high calorie estimates because when green crab invade at high densities, they are unlikely to have unlimited access to shellfish. That is, high densities of green crab invasion will reduce harvested shellfish biomass through predation, however if there is intraspecific competition for prey then high crab densities are likely to reduce the number of calories consumed per crab74. Thus, it is unlikely that the highest crab invasion densities will consume calories at the highest rates. The lower calorie estimates demonstrate a limited access scenario and therefore present a more realistic change in biomass. Yet, significant estimates of possible shellfish harvest loss are reached both at high calorie diets but medium crab densities, and at high crab densities but medium calorie diets, suggesting that there are multiple plausible invasion scenarios that could result in a loss to shellfish harvest.\n\nThe lack of previous data on economic loss to green crab makes it difficult to compare our estimates of value-at-risk in Puget Sound to losses experienced elsewhere. In the northeast Atlantic, the shellfish industry was estimated to have lost 4.5 million kgs of biomass and $22.6 million USD in revenue each year from green crab, though this estimate did not include oyster species, potentially underestimating total costs, and did not describe what proportion of the total fishery was lost to green crab38. Without knowing what the total biomass of this northeast Atlantic fishery is, it is not possible to compare the estimates of value-at-risk to green crab invasion as calculated in this study. This same study estimated the cost of green crab’s future predation impact in the northeast Pacific at $844,000 year-1 once green crab expand into Puget Sound and up to the Aleutian Islands38. This coarse estimate may be low as it does not include regional variation in feeding rate or all the harvest shellfish species at risk, though it does estimate costs across increasing invasion densities. However, total Washington State shellfish harvest value was underestimated as $17.2 million USD for the entire state (not including oysters)38, while in our study we demonstrated that harvest of hardshell clams and mussels in Puget Sound alone was worth $23.2 million USD. Additional value-at-risk estimates for regions already invaded by green crab would improve estimates for not yet invaded regions.\n\nRecreational shellfisheries in Puget Sound are also likely to be directly affected by loss of shellfish biomass. As number of recreational harvest days per year decreases, harvesters are likely to experience reduced shellfish harvest and loss of cultural benefits, such as family engagement and traditional harvesting by native coastal tribes. Indirect effects may result when local human communities located near harvest beaches experience reduced benefits as fewer harvesters buy supplies, permits, food and lodging. The social and environmental-engagement value of recreational shellfish harvest might be difficult to quantify but is an important aspect of harvest value that is often ignored75.\n\n\nAdditional ecosystem changes\n\nThe introduction of C. maenas is a threat to biodiversity and ecosystem function for Puget Sound’s near-shore food web23,38,39,76. The diet preference for bivalves and resulting ecological impact has been relatively similar across invaded regions37. Assuming this remains true for Puget Sound, removal of bivalves by green crab may have indirect effects on shorebird populations by removing their prey, as seen in other sites in the northeast Pacific37. Green crab bivalve predation also may result in a shift in the bivalve community if bivalve species that are less-preferred by green crab replace those consumed more heavily77. When this occurred in Bodega Harbor, California, green crab suppressed the native clam, Nutricola spp., which had an indirect positive effect on the nonnative clam, Gemma gemma. Green crab also have the potential to increase tertiary productivity in Puget Sound, because their larvae provide a prey resource to fish species15 and adults are likely to become prey for birds, seals, and fish that normally feed on local crab species.\n\nShellfish threatened by this invasion provide more than just commercial and recreational harvest revenue. By filtering toxins and nutrients from Puget Sound, shellfish help to increase oxygen levels and reduce toxin accumulation in other organisms. Eutrophication in Hood Canal and southern Puget Sound is likely to increase with a decline in shellfish populations78. Because these nonmonetary values are not incorporated in model estimates, this study likely underestimates the total value lost to green crab invasion.\n\n\nMotivating prevention and mitigation of invasive impacts\n\nManagers can prepare for major losses of harvest and revenue by initiating strong preventative measures3,13,25. Current efforts in Puget Sound to prevent green crab invasion include restricting out-of-state imports of shellfish, encouraging commercial shellfish harvesters to inspect their equipment before transferring gear between invaded and non-invaded regions, requiring ballast exchange before entering Puget Sound, and instituting a detection program (http://wdfw.wa.gov/ais/carcinus_maenas/) that incorporates community volunteer groups and paid specialists79. These efforts are useful but could be improved if more funding was allocated to preventative efforts. For example, funds could be used to better enforce gear inspection and ballast exchange. At present, ballast exchange is not required between Oregon, Washington, and BC, though green crab are already present along the outer coast of each of these states/provinces (Washington Department of Fish and Wldlife (WDFW) ballast water program)80,81. In addition, increasing the number of paid specialists sampling for green crab would increase the chances of catching green crab invasion early and prevent further spread in Puget Sound82.\n\nDespite current efforts to limit human introduction of green crab into Puget Sound, climate change resulting in warming sea waters and changing current flow will likely result in larval transport into Puget Sound without further human assistance34,83. Plans to mitigate the impact of green crab will be most effective if they are in place before invasion occurs. Managers can prepare commercial harvesters to take preemptive measures in reducing the green crab’s impact by altering their current methods of shellfish aquaculture. Oyster and mussel racks suspended above the bottom substrate may limit green crab’s access to commercially cultured bivalves and this is already a common practice in neighboring BC (BC Shellfish Grower's Association Shellfish industry encyclopedia)84. Anti-predator netting has been used successfully in the northeast Atlantic, reducing loss of clam biomass to predation by 13–55%85. Netting may be used to minimized predation on Puget Sound clams and has been tested in BC, though this method has detrimental side-effects on other infaunal species also netted in the mudflat86. A comprehensive post-invasion plan would combine measures to mitigate impacts with monitoring and control efforts25,87.\n\n\nIncorporating uncertainty of future invasion impacts\n\nFuture research using the consumption model presented in this study can improve upon these estimates of green crab impact by incorporating greater detail on predation rates, shellfish recruitment effects, and estimates of total biomass of shellfish in Puget Sound. The impact on each shellfish species could take into account both green crab preference for these prey species, their probability of encountering each species in the field, and density effects on predation rates. Future estimates may also benefit from considering the decrease in predation rate as total shellfish biomass in all of Puget Sound declines due to biomass loss and reduced recruitment of juvenile shellfish. In general, estimates of green crab invasion densities would be improved if future studies that sample existing populations of green crab included crab densities measured in a metric that is repeatable across regions, such as crab density per square meter measured using dredges or quadrats. While CPUE is useful for comparing densities within and between regions in a single study, measurements are difficult to translate into a density of crab per area and results may vary with trap type and deployment methods used.\n\nIt is not possible to precisely estimate the invasion impacts of green crab and other invading species because of temporal and spatial variation across invaded regions26–29 and the effect of management responses aimed at mitigating those impacts. However, though few data are available about specific impacts before invasion, researchers can refine estimates of value-at-risk by incorporating a range of potential invasion parameters: density of individuals, number of arrivals, potential predation and competition interactions, and economic impacts, these general estimates can produce more accurate assessments of what is known regarding the invading species12,25. If estimates are too simplistic and do not include uncertainty measures or are not made at all there is little motivation to rationalize economic spending to monitor, prevent or mitigate for species invasions before invasion occurs19.\n\n\nConclusions\n\nAt high densities or high calorie predation rates green crab may have the potential to reduce revenue from shellfish harvest and processing by as much as $1.6–41 million USD, representing real uncertainty in possible impacts. Value lost to shellfish harvest is dependent on the density of crabs that invade harvest areas and the actual calories consumed by each crab, parameters that are likely to be a product of the individual rate of predation and the accessibility to prey. Loss of shellfish has implications for recreational shellfish harvest and the potential for reduced filtration rates that may lead to increased eutrophication in already threatened coastal habitats88. Preventing or reducing the effect of high-impact species invasions should be a priority as these invasions have direct economic impacts and a range of indirect effects on ecosystem function (Puget Sound Partnership action agenda)89,90. By incorporating uncertainty when estimating impacts from invasions, management plans can describe the range of potential costs of invasion and motivate preventative action in order to prepare for future ecosystem damage even when local impacts are still unknown12,13,25,87.", "appendix": "Author contributions\n\n\n\nMM and KC conceived the study. MM designed the consumption model and conducted analyses. MM prepared the first draft of the manuscript. MM and KC contributed to preparation of the manuscript, both were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nMM was funded by the Canadian Aquatic Invasive Species Network (CAISN) and an NSERC Discovery Grant (# 06-5566). KC was funded by the Canada Research Chairs program. Both KC and MM were funded by the Canadian Foundation for Innovation (# F07-0010).\n\n\nAcknowledgements\n\nWe would like to thank the following scientists for their help and expertise: Dr Robert Ahrens, University of British Columbia, provided expertise in developing the model and sensitivity analysis; Mark Plummer, National Oceanic and Atmospheric Administration, provided shellfish aquaculture and shellfish harvest area data for Puget Sound; Scott Kellogg, Washington State Department of Health, provided 2010 commercial shellfish growing area ArcGIS data; and Jon Bridgman, Puget Sound Partnership, provided Puget Sound Action Areas ArcGIS data.\n\n\nReferences\n\nLodge DM, Williams S, MacIsaac HJ, et al.: Biological invasions: recommendations for U.S. policy and management. Ecol Appl. 2006; 16(6): 2035–2054. PubMed Abstract \| Publisher Full Text\n\nMillennium Ecosystem Assessment: Ecosystems and human well-being: synthesis. 137 (Island Press, Washington, D.C., 2005). 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Publisher Full Text\n\nBC Shellfish Grower’s Association: Shellfish, industry encyclopedia. 2012. Reference Source\n\nBeal BF, Kraus MG: Interactive effects of initial size, stocking density, and type of predator deterrent netting on survival and growth of cultured juveniles of the soft-shell clam, Myaarenaria L., in eastern Maine. Aquaculture. 2002; 208(1–2): 81–111. Publisher Full Text\n\nMunroe D, McKinley RS: Effect of predator netting on recruitment and growth of manila clams (Venerupis philippinarum) on soft substrate intertidal plots in British Columbia, Canada. J Shellfish Res. 2007; 26(4): 1035–1044. Publisher Full Text\n\nHoran RD, Perrings C, Lupi F, et al.: Biological pollution prevention strategies under ignorance: the case of invasive species. Am J Agric Econ. 2002; 84(5): 1303–1310. Publisher Full Text\n\nOfficer CB, Smayda TJ, Mann R: Benthic filter feeding: a natural eutrophication control. Mar Ecol Prog Ser. 1982; 9: 203–210. Publisher Full Text\n\nHedgpeth JW: Foreign invaders. Science. 1993; 261(5117): 34–35. PubMed Abstract \| Publisher Full Text\n\nPuget Sound Partnership. Action agenda. 2008. Reference Source" }	[ { "id": "848", "date": "18 Mar 2013", "name": "Ryan Chisholm", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript looks at the potential economic impacts of invasive European green crabs on the shellfish industry in Puget Sound. They come up with a range of dollar values for potential economic impacts, based on both economic and ecological underlying uncertainties.The manuscript is mostly sound, although I have one concern relating to their treatment of “primary” and “secondary” values:•\n\nHow are “Processing margin” and “Distribution margin”, which make up the secondary value in Table 2, calculated? I understand that you are just applying ratios from ref. 72, but I want to understand where these ratios come from (I looked at ref. 72 briefly, but it wasn’t easy to figure out). Does this secondary value represent the difference between the market value and harvested value of the fish? In any case, you must be careful to avoid double-counting when you sum the primary and secondary values. Suppose someone catches some fish and sells the unprocessed fish to me for $100 and then I employ someone to process the fish for $20, and then I sell the fish for $150. How much total value has been added to the economy here? The answer is less than $150, because the buyer of the finished product would otherwise have just spent his $150 elsewhere (although presumably he would not have derived as much utility from the alternative purchase—thus his decision to spend his money on my fish when the option arose). Equally, the person I hired for $20 would have been hired somewhere else had I not hired him (although presumably for less than $20, otherwise he would not have accepted my job offer).The upshot of this is that, in your paper, you should avoid adding the primary and secondary values together, or alternatively you should find a good reference to say that adding them is OK (contrary to what I have said above). It’s possible that I’ve misunderstood what you’ve done (e.g., maybe in what you describe as the “Processing Margin”, you or the references you cite are somehow accounting for the issues I outline above), in which case you just need to explain it a bit better.Other comments:•\n\nFigure 1 legend: “Figure and Maxtent potential distribution model”. Do you mean “MaxEnt”?•\n\np6: “the following simple linear model” Prefer just “the following simple model”, because “linear model” will make most readers think of linear regression models, which is not what you are doing.•\n\np9 and throughout: “kgs” -> “kg”", "responses": [ { "c_id": "722", "date": "28 Feb 2014", "name": "Megan Mach", "role": "Author Response", "response": "Dear Dr. Chisholm,Thank you for taking the time to review our article, we believe it has improved because of your review. I have included our responses and notes on the changes made to the manuscript (in normal font) as a result of your comments (denoted in italics).\"How are “Processing margin” and “Distribution margin”, which make up the secondary value in Table 2, calculated?\"To clarify how processing and distribution margin were calculated we have added specific text to Table 2. This information should not only clarify these two metrics but lay out more clearly how each of the values in the table were calculated. We specifically describe how these were calculated as part of our response to the following comment. \"Does this secondary value represent the difference between the market value and harvested value of the fish?\"The processing margin (one of the secondary values) represents the difference between the wholesale market value and the harvest value, however the distribution margin is in addition to wholesale value (15% of processed value, described more below).Revisiting the original documentation for these values (GS Gislason & Associates Ltd., 2007) we caught an error in our processing and distribution values. We had calculated processing margin of shellfish based on the Total fisheries processing margin, however, these values should have been calculated as the difference between Wholesale Value and Landed Value for all shellfish (from both capture and aquaculture, page 12 of GS Gislason & Associates Ltd., 2007). We have corrected the processing margin accordingly for BC in Table 2, from $100m to $71m. This then changes the distribution margin, which is 15% of harvesting + processing margin (the processed value; page 14 of GS Gislason & Associates Ltd., 2007). All relationships are now described as footnotes to Table 2 to ensure the calculations are transparent, including where in the original reference on British Columbia the values originated.In addition, the % values in parentheses at the top of each column of Table 2, representing percent value change at increasing crab densities and calories consumed, had been changed but the value in the table had not. This is now corrected. These percentages were calculated from the values in Table 3 as the percent change from the original harvest value to each of the low, medium, and high densities, and low, medium, high calorie diets. Because other values in the table were dependent on these percentages, there was a small shift in the processing margin, labour income, and employment when the percentage was corrected. These value shifts were small and in no way changed the overall message of the table. \"Remove references to a “Total” harvesting, processing and distribution margin, as this total does not consider the possibility of underselling the estimated values.\"We have removed references to “total” value that include harvesting, processing and distribution from the text as well as from Table 2. Additional comments\"Figure 1 legend: “Figure and Maxtent potential distribution model”. Do you mean “MaxEnt”?\"Yes, thank you, this has now been corrected. \"p6: “the following simple linear model” Prefer just “the following simple model”, because “linear model” will make most readers think of linear regression models, which is not what you are doing.\"We have changed the wording to ensure we do not misrepresent our model. \"p9 and throughout: “kgs” -> “kg”\"This has been corrected throughout the manuscript." } ] }, { "id": "2041", "date": "09 Oct 2013", "name": "Nicholas Bax", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI personally have some level of discomfort with looking at the risks of invasions (or other environmental problems) solely from the point of view of consequences (called economic value at risk here) without also considering likelihood. This has been addressed to some degree in the introduction, but I think needs to be further emphasized both in the introduction and the discussion/conclusions. One reason for including likelihood is that it reduces the danger that a result can be considered alarmist and dismissed. The well-known human failings in assessing risk by focusing on consequences rather than likelihood is very well discussed in the literature. Examples include the risk of a nuclear accident being perceived as of greater risk to an individual’s health than smoking. A second reason is that including probabilities moves the assessment from qualitative, or semi-quantitative (as in this case) to fully quantitative, and this tends to require a more systematic consideration of the problem.\n\nThere are two major areas where I think the papers needs modifying, one of which, if I have interpreted the methods correctly, would reduce estimated losses by about 2/3: There is no discussion of earlier estimates of the impacts of the green crab on Puget Sound shellfish (and other) resources – e.g. Lafferty and Kuris, 1996 (Ecology), or costs of marine invasives in general (e.g. Williams and Grosholz, 2008 (Estuaries and Coasts).) The assumption that green crab would have a diet of 60-100% harvestable shellfish, is not well supported by any literature provided, but will directly affect the conclusions. The approach taken of simply adding the predation estimates for each species together is not realistic. One could equally argue that they are interchangeable and that shellfish (e.g. because of handling time?) would never form more of the diet than that represented by one species. At the individual crab level, each crab would have to move between beds or farms of the three species every day. If they did not, and stayed in one area as seems likely, they would only have the opportunity to eat one of the three shellfish species each day (week? month? year?). So I believe that adding them together is unrealistic.I would also like to see the following points addressed:It is assumed that a green crab invasion would be uniform, through time and space, in Puget Sound. How realistic is this? There seems to be no allowance for the influence of predator-prey size ratios on the ability of green crabs to attack their prey. What effect will this have? Is there a size beyond which shellfish are protected? Could rearing practices reduce the impact of predation? What is the impact of reduced prey numbers on the growth and survival rate of the remaining shellfish? What is the impact of the green crab on reducing existing predation from crabs that it will displace? There is no discussion of price elasticity in supply. Would prices of shellfish stay the same if supply dropped by 60% as suggested? I did not note any discussion of expenses. Net profit would be a more informative indicator than revenue. The numbers in Table 2 for direct loss of shellfish (0.2 to 55.9%) do not seem to agree with those in Table 3 (0.3 to 68.7%), or in those in the text describing the results.", "responses": [ { "c_id": "721", "date": "28 Feb 2014", "name": "Megan Mach", "role": "Author Response", "response": "Dr. Bax,Thank you for taking the time to review our article, we believe it is much improved by your comments. I have included our responses and notes on the changes made to the manuscript (in normal font) as a result of your comments (denoted in italics).\"In response to comments regarding additional discussion of likelihood in the introduction and discussion:\"We agree that the issue of likelihood is critical, which is why we consider such wide ranges of values across several parameters. We include both high and low estimates of green crab consumption that account for a broad range in calorie intake levels and invasion densities to demonstrate the huge uncertainty inherent in how green crab will interact and impact this new ecosystem. The literature was not sufficient to inform explicit likelihood values for each of these scenarios, so we simply explore a wide range and report results across that range. Thus, issues of likelihood are implicit in the structure of our entire analysis, just without the specification of relatively likelihood. For example, under scenarios where few green crab invade, their impacts are likely to remain relatively low (depending on calorie consumption estimates).We also discuss some aspects of likelihood in the introduction as well as the discussion. For example, in the discussion we describe green crab as an opportunistic feeder likely to switch species when abundances become to low and that intraspecific competition is likely to reduce calories consumed by each crab (page 15). \"There is no discussion of earlier estimates of the impacts of the green crab on Puget Sound shellfish (and other) resources – e.g. Lafferty and Kuris, 1996 (Ecology), or costs of marine invasives in general (e.g. Williams and Grosholz, 2008 (Estuaries and Coasts).)\"On page 16 we discuss earlier estimates of green crab’s future predation impact in Puget Sound and up to the Aleutian Islands; estimates are likely low because they do not include any regional variation in feeding rate or include many of the harvested shellfish species at risk (Lovell et al. ,2007). This same report underestimated Washington’s shellfish harvest value by almost 5 million USD when compared to the data used in our analysis.We did not include estimates of green crabs’ impact on annual value of oysters by Lafferty and Kuris (1996; Table 1 of their article) because their estimates include Oregon, the outer coast of Washington and Puget Sound. We did not have a way to estimate the Puget Sound threatened annual value alone. As an example of the results in the two studies--the Lafferty and Kuris study estimates that green crab will reduce oyster value by $20 mil in 1990 USD (~$33 million in 2009 USD) for Oregon and Washington, while we estimate total revenue in 2009 for oysters in Puget Sound alone to be ~$14 million.I have removed the freshwater Zebra mussel example and replaced it with a jellyfish example from the review by Williams and Grosholz (2008) to provide a marine example of an economically costly invasive species (first paragraph of the introduction, page 3). \"The assumption that green crab would have a diet of 60-100% harvestable shellfish, is not well supported by any literature provided, but will directly affect the conclusions. The approach taken of simply adding the predation estimates for each species together is not realistic.\"This estimate allows for other species to comprise up to 40% of C. maenas’s diet and that crabs can feed on any of the three shellfish species we are modeling (mussels, clams, & oysters). These estimates are supported by a study (Grosholz and Ruiz, 1996) that found these shellfish species groups as ranging from 20 to 35% of the total green crab diet. Our model assumes that green crab would have a diet that is made up of 60-100% of the harvestable shellfish (all three species together) over the course of a year based on this study, as described under Diet in the Methods section on page 9.\"One could equally argue that they are interchangeable and that shellfish (e.g. because of handling time?) would never form more of the diet than that represented by one species. At the individual crab level, each crab would have to move between beds or farms of the three species every day. If they did not, and stayed in one area as seems likely, they would only have the opportunity to eat one of the three shellfish species each day (week? month? year?). So I believe that adding them together is unrealistic.\"This model estimates all calories consumed by the range of densities of green crab in harvestable areas over the course of a year, assuming a range of calorie requirements of the crabs. It does not assume that individual crabs move across beds every day, but rather we estimate the entire invading population (in harvest areas). \"It is assumed that a green crab invasion would be uniform, through time and space, in Puget Sound. How realistic is this?\"Treatment of invasion as uniform across time and space is a limitation of this model. We have made a point to only discuss regions that have data on harvest densities, and only discuss impacts to harvest value (in commercial shellfish harvest areas) to limit the spatial scale to which our model applies. We describe the huge variability in past invasions that make it difficult to assess how green crab invasion will impact Puget Sound shellfish harvest (page 19), this is another reason we incorporated such a broad range of possible values, and we have also added additional text under “Incorporating uncertainty in future invasion impacts” on page 19.\"Future research using the consumption model presented in this study can improve upon these estimates of green crab impact by incorporating greater detail on …the variation of predation rate and calorie intake over time, spatial differences in invasion effects…” \"There seems to be no allowance for the influence of predator-prey size ratios on the ability of green crabs to attack their prey. What effect will this have? Is there a size beyond which shellfish are protected? Could rearing practices reduce the impact of predation?\"On page 9, where we discuss the number of calories consumed by each adult green crab (Cal) we discuss green crabs preference for juvenile shellfish and that the harvest of shellfish is calculated for adult shellfish. These estimates may result in overestimating predation impacts.Rearing practices could indeed reduce the impact of green crabs on juveniles. This is discussed at the end of this same paragraph on page 9 along with a discussion of how plastic behavior, such as burrowing depth may also reduce predation. We also discuss rearing practices on page 18-19 in the “motivating prevention and mitigation of invasive impacts” section. \"What is the impact of reduced prey numbers on the growth and survival rate of the remaining shellfish?\"On page 15 we discuss how “the reduction of shellfish biomass [] has the potential to limit recruitment of shellfish by reducing the population of reproductive adult shellfish and by preying on newly settled juvenile shellfish.”We have added “The reduction in shellfish biomass may also increase the growth and survival of juvenile shellfish by reducing competition for space and food” to this same section.In addition, in the section “additional ecosystem changes” on page 17 we discuss that the ecological impact of green crab has been relatively similar across regions. Assuming this remains true for Puget Sound, the impact of a reduction in prey number may result in a shift in the bivalve community if a bivalve species that is less preferred by green crab replaces those consumed more heavily. \"What is the impact of the green crab on reducing existing predation from crabs that it will displace?\"The impact of green crab on native crab likely includes 1) reduced prey availability through competition; 2) reduced native crab biomass through predation (discussed briefly on page 5) and competition for shelter; and 3) reduced biomass through loss of nursery habitats (discussed on page 6).We added additional discussion of the potential indirect impact of green crab on native crabs to page 17 under “Additional ecosystem changes.” There will likely also be an indirect impact on shorebirds (also discussed here). Reduced native crab biomass could potentially affect our estimates of the impacts of green crab as native crabs will remove less shellfish biomass from the harvest areas. We have added a comment to this effect on page 17. \"There is no discussion of price elasticity in supply. Would prices of shellfish stay the same if supply dropped by 60% as suggested?\"We have now included a discussion (page 16) of change in supply. This is an excellent point.“Not included in our estimates is a prediction of how change in shellfish biomass supply will affect willingness to pay for those shellfish (‘price elasticity’). If there is a strong ‘local’ aspect to the demand (a greater willingness to pay for Puget Sound shellfish), reductions in shellfish harvest of 60% would likely trigger an increase in market price. However, it is also likely that as costs increase, consumers will choose to eat shellfish produced in other regions that continue to produce low costing shellfish.” \"I did not note any discussion of expenses. Net profit would be a more informative indicator than revenue.\"Both metrics appear to be of interest to different parties. For example, policymakers and politicians may be more concerned with revenues, because many costs are expenditures that fuel the local economy (especially wages, which are typically a large share of costs in more-developed countries). Unfortunately, as we do not have access to the costs at each stage (primary or secondary economic value), we had no real basis to estimate profits. We added a discussion to this effect on page 12. \"The numbers in Table 2 for direct loss of shellfish (0.2 to 55.9%) do not seem to agree with those in Table 3 (0.3 to 68.7%), or in those in the text describing the results.\"The percent loss in Table 3 was specific to the total shellfish biomass under various scenarios as compared to the baseline biomass. The percent loss in Table 2 is the total shellfish revenue value under various scenarios as compared to the baseline revenue. To make this less confusing I have removed the percent loss values from Table 3 and altered the text in the table legend of Table 2." } ] } ]	1	https://f1000research.com/articles/2-66
https://f1000research.com/articles/3-94/v1	23 Apr 14	{ "type": "Opinion Article", "title": "Data publication consensus and controversies", "authors": [ "John Kratz", "Carly Strasser", "Carly Strasser" ], "abstract": "The movement to bring datasets into the scholarly record as first class research products (validated, preserved, cited, and credited) has been inching forward for some time, but now the pace is quickening. As data publication venues proliferate, significant debate continues over formats, processes, and terminology. Here, we present an overview of data publication initiatives underway and the current conversation, highlighting points of consensus and issues still in contention. Data publication implementations differ in a variety of factors, including the kind of documentation, the location of the documentation relative to the data, and how the data is validated. Publishers may present the data as supplemental material to a journal article, with a descriptive “data paper,” or independently. Complicating the situation, different initiatives and communities use the same terms to refer distinct but overlapping concepts. For instance, the term “published” means that the data is publicly available and citable to virtually everyone, but it may or may not imply that the data has been peer-reviewed. In turn, what is meant by data peer review is far from defined; standards and processes encompass the full range employed in reviewing the literature, plus some novel variations. Basic data citation is a point of consensus, but the general agreement on the core elements of a dataset citation frays if the data is dynamic or part of a larger set. Even as data publication is being defined, some are looking past publication to other metaphors, notably “data as software,” for solutions to the more stubborn problems.", "keywords": [ "The idea that researchers should share data to advance knowledge and promote the common good is an old one", "but in recent years the conversation has shifted from sharing data to “publishing” data1–3. This shift in language stems from the conviction that datasets should join the scholarly record and be afforded the same first class status as traditional research products like journal articles4. While many in the scholarly communication community share this goal", "different people and organizations often imply different things by the phrase data publication." ], "content": "Introduction: what does data publication mean?\n\nThe idea that researchers should share data to advance knowledge and promote the common good is an old one, but in recent years the conversation has shifted from sharing data to “publishing” data1–3. This shift in language stems from the conviction that datasets should join the scholarly record and be afforded the same first class status as traditional research products like journal articles4. While many in the scholarly communication community share this goal, different people and organizations often imply different things by the phrase data publication.\n\nThe community largely agrees on two essential properties of a data publication2,4. First, published data is publicly available now and for the indefinite future; access might demand payment of fees or acceptance of a legal agreement, but not the approval of the author. Second, like a book or journal article, a data publication can be formally cited. Open questions flock around a third property: how and to what extent a published dataset must be validated. In an effort to clarify the terminology, Callaghan et al. (2012)4 draw a distinction between data that has been shared, published (lower-case “p”), or Published (upper-case “P”): shared data is available, published data is available and citable, and Published data is available, citable, and validated. In practice, availability is usually satisfied by depositing the dataset in a repository, citability by assigning a persistent identifier (e.g. a Digital Object Identifier, or DOI), and validity by peer review.\n\nThe underlying goals of data publication are to enable research to be reproduced and data to be reused. Hidden primary data exacerbates science’s very public “reproducibility crisis”5–9, most recently illustrated by the collapse of a pair of irreproducible Nature articles describing a simple method to transform somatic cells into pluripotent stem cells10,11. Widespread publication of the data underlying research papers could help expose both honest errors and fraud12. The leaders of the US National Institutes of Health (NIH) recently cited “provid[ing] greater transparency of the data that are the basis of published manuscripts” as one way to improve scientific reproducibility13.\n\nJournals already frequently require authors to supply underlying data on request. In 2011, Alsheikh-Ali et al.14 found that 88% of high-impact journals required a statement regarding the availability of underlying data; half of those made willingness to provide data a condition of publication. However, the authors of 59% of papers examined in the study failed to adhere to the availability instructions. Vines et al. (2014)15 could only obtain underlying data from 101 of 516 papers published from 1991 to 2011. Availability dropped off sharply with time; data could be obtained from only two of the 62 oldest papers. Now, some journals require that underlying data be published simultaneously with the article.\n\nIn 2010, a coalition of Ecology and Evolutionary Biology journals began to require that the data underlying articles be archived with a maximum embargo of one year16,17. F1000Research has had a similar policy (without an embargo period) since its inception, and the Public Library of Science (PLOS) journals followed suit earlier this year18. Although there can be no substitute for funding new experiments and data collection, appropriate data reuse lowers costs and accelerates research. Documenting, publishing, and archiving data is time consuming and costly, but usually far less so than repeating the data collection. For example, Open Context published archaeological data from a site in eastern Turkey at the substantial cost of $10,000–15,0000, but this publication expense was minor compared to $800,000 spent to collect the data19. Piwowar (2011) contrasted the impact of $100,000 in National Science Foundation (NSF) grants, which generates an average of three to four papers, with an estimate that the same investment in curating, archiving, and publishing data could contribute to over 1,000 publications20. Furthermore, while some data is merely expensive to recreate, time-dependent or ephemeral data, (e.g. climate records or observations of unique astronomical events) should be published because it can never be recreated for any price21.\n\n\nTypes of data publication\n\nThe still-congealing phrase “data publication” covers diverse classes of research objects published via diverse processes. Depending on the speaker, a data publication might be a spreadsheet on a website, a set of images in an institutional archive, a stream of readings from a weather station transmitted over the internet, or a peer-reviewed article describing a dataset. Because disciplines, sub-disciplines, and individual researchers consider different assortments of digital material to be data, it is unlikely that any single structure will suit every discipline and dataset. But, we can hope that a manageable number of designs will fit most data. Five data publication models described by Lawrence et al. (2011) are distinguished “by how the roles involved in publication are distributed between the various actors” (e.g. the author, archive or journal)3. Here, we will more simply group data publications into three categories based on the accompanying documentation; a dataset may supplement a traditional research paper, be the subject of a “data paper”, or be independent of any paper (Figure 1).\n\nSome, but not all, publishers review datasets to validate them.\n\nThe most familiar kind of data publication is a traditional journal article accompanied by underlying data. That data can be hosted by the journal as supplementary material or deposited in a third-party repository. The trend is away from supplemental material because repositories are considered to be better suited to ensure long-term preservation and access to the data. For instance, The Journal of Neuroscience stopped publishing supplemental material in 2010; the announcement promotes disciplinary repositories as “vastly superior to supplemental material as a mechanism for disseminating data”22. Data underlying any peer-reviewed or otherwise “reputable” publication can be deposited in the Dryad repository. Dryad makes data available and citable, but the publisher of the article must manage any assessment of scientific validity. Other third-party repositories include Figshare, Zenodo, institutional repositories (e.g. the Purdue Research Repository), and discipline-specific repositories (e.g. DNA sequences are deposited in GenBank23 and protein structures in the Protein Data Bank24).\n\nA data paper describes a dataset with thoroughly detailed rationale and collection methods, but lacks any analysis or conclusions25. Data papers are flourishing as a new article type in journals such as F1000Research, Internet Archaeology, and GigaScience, as well as in dedicated journals like Geoscience Data Journal, Nature Publishing Group’s Scientific Data, and a trio of “metajournals” from Ubiquity Press.\n\nData paper length and structure varies between journals, but the tendency is toward a short, tightly structured format. All journals require an abstract, collection methods, and a description of the dataset; a few encourage authors to suggest potential uses for the data (e.g. Internet Archaeology, and Open Health Data). Some journals supplement this general framework with field-specific sections. (e.g. Internet Archaeology and the Journal of Open Archaeology Data each include a section for temporal and geographic scope.) Data papers are most sharply defined not by the presence of any particular information, but by the absence of analysis or conclusions. A crisp distinction from other article types is important because many journals do not consider a data paper to be prior publication if the authors seek to publish an analysis of the same dataset (e.g. Nature-titled journals, Science, and others listed by F1000Research).\n\nData journals generally limit themselves to publishing the description of the dataset; a trusted repository publishes the data itself. For instance, Scientific Data and Geoscience Data Journal each direct authors to a list of approved repositories. As an exception, GigaScience hosts data in an integrated repository named GigaDB. An early implementer of data papers, The International Journal of Robotics Research25 is unusual in that they permit authors to host datasets on their own websites.\n\nTo be useful or reproducible, a dataset must be accompanied by descriptive information (i.e. metadata)21, but this need not take the form of a journal article. Instead, some repositories publish rich, structured and/or freeform description together with the data. The distinction between a data repository and a data publisher is often indistinct. Repositories provide access and citability, but the degree of validation varies widely and few are equipped to provide peer review. For instance, to make data publication as easy as possible for authors, Figshare and Zenodo publish datasets from any field with minimal validation.\n\n\nAvailability\n\nFundamentally, to publish is to make public, and to publish data is to make data publicly available. Present availability requires mechanisms for access; future availability also requires preservation (e.g. long-term storage, format migration)21,26,27. As in print publication, published data need not be free or legally unencumbered, and data use agreements constrain many published datasets. If access is limited, it should be contingent on clear and objective criteria; writing a request to the creator for permission should not be part of the process. For example, before granting access to restricted data, The Interuniversity Consortium for Political and Social Research (ICPSR) judges the applicant’s proposed security measures, but not the merit of their research. Datasets from social science or clinical studies that involve human participants are easily the most common source of access restrictions because of the need to protect privacy. In the United States, the Health Insurance Portability and Accountability Act of 1996 (HIPPA) Privacy Rule severely limits the disclosure of medical information28.\n\nAs a practical matter, publishing a dataset usually includes depositing it in a trustworthy repository. What constitutes a “trustworthy” repository is somewhat subjective and there are a handful of certification schemes to choose from. In 2007, The Center for Research Libraries (CRL) published the most extensive scheme: the Trusted Repository Audit Checklist (TRAC)29. Many repositories consult TRAC for self-assessment, but only four (listed by the CRL) have completed the lengthy and rigorous process to be officially certified. The process to obtain a Data Seal of Approval (DSA) is considerably more streamlined. The DSA guidelines were also first released, by The Dutch Data Archiving and Networked Services (DANS), in 2007; 24 repositories have been stamped with the DSA since then. Few of the hundreds of repositories in operation (e.g, the 973 now listed Databib or the 609 at re3data.org) have pursued any kind of certification. Given the low adoption of repository certification, a more typical way to decide trustworthiness is to judge by the organization responsible. Repositories run by governments or large universities are likely to be considered trustworthy (although the effects of the 2013 US government shutdown on the PubMed biomedical article database30 might give one pause).\n\n\nCitability\n\nData citation is the element of publication that has come the farthest toward consensus. This year, a coalition–including Future Of Research Communication and E-Scholarship (FORCE11)31, the Committee on Data for Science and Technology (CODATA)32, and the Digital Curation Centre (DCC)–released a Joint Declaration of Data Citation Principles. The first of the eight principles states, in part, that “[d]ata citations should be accorded the same importance in the scholarly record as citations of other research objects, such as publications”. Most of the time, this means that when a published dataset contributes to a paper, it should be cited formally in the reference list.\n\nData publishers enable formal citation by assigning unique permanent identifiers, most commonly the same ones used for journal articles: Digital Object Identifiers (DOIs). In addition to clarifying exactly what resource is being cited, a DOI can be resolved to locate the referenced dataset. Note, however, that a DOI is neither sufficient nor necessary for citability- if a dataset moves and the DOI is not updated, the citation breaks and, conversely a well-maintained web-address works as well as a DOI.\n\nThe present consensus is that a dataset should be cited using, at a minimum, five elements largely familiar from article citations: creator(s), title, year, publisher and identifier. This format agrees with CODATA’s recommendation32 and conveys all the information required to obtain a DataCite DOI33 or be listed in the Thomson-Reuters Data Citation Index. However, this article-derived format fails to address some of the complications unique to datasets, described below.\n\nThe first major complication that data citation faces is the need for deep citation. When supporting an assertion in writing, it usually suffices to cite the entirety of a journal article and leave it to the inquisitive reader to find the relevant passage. But, to reproduce an analysis performed on a subset of a larger dataset, the reader needs to know exactly what subset was used (e.g. a limited range of dates, only the adult subjects, wind speed but not direction). Datasets vary so widely in structure that there may not be a good general solution for describing subsets. The most common suggestion is to cite the entire dataset in the reference list and describe the subset in the text of the paper34. In straightforward cases, the Federation of Earth Science Information Partners (ESIP) and the National Snow and Ice Data Center (NSIDC) both recommend including a list of variables or range of dates in the formal citation.\n\nThe second major complication arises when datasets change. In the past, the printing process cemented one version of an article as the version of record. Even for traditional scholarly literature, web-based publishing and preprint servers (e.g. arXiv.org) are complicating the situation, but datasets are especially prone to be dynamic. Two kinds of dynamic datasets warrant consideration: growing datasets that add new data while never changing or deleting existing data, and revisable datasets where data may by added, deleted, or changed.\n\nConsider USC00046336, a weather station at the Oakland Museum. Each day, the high temperature, low temperature and amount of precipitation recorded at the Museum35 flow, together with data from more than 20,000 other stations, into the swelling Global Historical Climate Network (GHCN)-Daily36 dataset. Or, consider WormBase37, a genome database used by the Caenorhabditis elegans research community. WormBase encompasses genomic sequences of C. elegans and 20 related species massively annotated with gene structures, protein sequences, expression patterns, and a host of other information from empirical data and computational predictions. Every two months, WormBase responds to new data and better computational models by issuing a revised version with new material added and inaccurate material deleted or corrected.\n\nAdditions and updates to published datasets are extremely valuable, but a researcher seeking to reproduce an analysis of a dynamic dataset needs access to a particular version. To enable that access, previous versions must be preserved and citable. Growing datasets can be cited with an access date or a date range in the citation, as recommended by ESIP and NSIDC. Revisable datasets are more difficult; the most common approach is to accumulate revisions and periodically publish a new version with a citable version number. For example, WormBase identifies each release with a citable version number and makes all of the previous versions available.\n\nControversy persists around the specific issue of identifiers for dynamic datasets. DataCite recommends, but does not insist, that their DOIs refer to immutable objects. NSICD and ESIP instruct researchers to use a single identifier for growing datasets and include the access date in the citation; each major version of a revisable datasets gets a new identifier, but minor versions do not. In contrast, the DCC, Dataverse, and the UK Natural Environment Research Council (NERC) insist that any change to a dataset should trigger a new identifier4,34,38. To handle the difficulties with dynamic data that this policy creates, the DCC recommends periodically issuing growing datasets a new identifier that refers to the “time-slice” of new records and freezing versions of revisable datasets as individually-identified “snapshots”.\n\nThe difficulties surrounding deep citation and dynamic data could potentially be solved by turning the identifier-issuing process on its head. Instead of the dataset publisher issuing identifiers for data at the level that researchers seem likely to cite, researchers could issue identifiers for precisely the part of the dataset that they want to cite. The Research Data Alliance (RDA) Data Citation Working Group recently put forth a sophisticated proposal applicable to data in (or convertible to) databases. Identifiers created under this scheme would wrap together identification of a database, a query to return the cited dataset, the version of the database queried for this analysis, and a number of other useful components. Although promising, many technical and policy issues must be resolved before this approach can be widely adopted.\n\n\nValidation\n\nData validation is the least resolved aspect of data publication, and fundamental questions are still unanswered: What minimum level of quality should a published dataset guarantee? How and by what criteria can datasets be evaluated against that guarantee? Is literature peer review an appropriate model?\n\nCallaghan et al. (2012)4 draw a useful distinction between technical and scientific review. Technical review verifies that a dataset is complete, its description is complete, and that the two match up. Domain expertise is generally not required, and many repositories provide at least some level of technical review. Scientific review evaluates the methods of data collection, the overall plausibility of the data, and the likely reuse value. Scientific review does require domain expertise, making this level of validation more difficult to organize, and few repositories provide it. When data is published with a data paper, review may be split between the repository for technical review and the data journal for scientific review.\n\nPeer review guarantees that journal articles entering the scholarly record reach some level of validity (although the aforementioned reproducibility crisis calls into question exactly what that level is). In many fields, peer-reviewed publications enjoy a much higher status than any other literature. Any effort to apply the prestige of “publication” to datasets cascades naturally into an effort to apply the prestige of “peer review”. But as data validation seeks to model itself on literature peer review, literature peer review itself is in flux39–41. Open peer review at F1000Research and post-publication commenting at PubMed Commons are just two of many ongoing web-enabled experiments in article evaluation.\n\nJournal article reviewers traditionally consider whether the methods used are appropriate for the questions asked and the data collected support the conclusions drawn. In the absence of particular questions and conclusions, it is not obvious what peer review of data should certify. A dataset may be suitable for some purposes, but not for others42. In addition, while a reviewer can be expected to read an entire article, they cannot inspect every point in a large dataset. Finally, researchers are already over-whelmed by peer review of articles43 and may find any increased workload unreasonable. Despite all these difficulties, venues for peer-reviewed data papers are opening rapidly.\n\nData paper journals wrap scientific peer review of the paper and the dataset together into a single process. GigaScience, an exception, assigns technical review of the dataset to a separate data reviewer. The standards that various data journals provide to reviewers are fairly uniform, with the exception that about half of consider novelty or potential impact, while the rest only require that the dataset be scientifically sound. While review standards are similar, processes differ widely.\n\nAs an example, compare Biodiversity Journal and Scientific Data. Both journals divide reviewer guidelines into three sections along similar lines, which Biodiversity Journal calls “quality of the data”, “quality of the description”, and “consistency between manuscript and data”. Scientific Data follows a traditional peer-review process: an editor appoints reviewers who are encouraged to remain anonymous. In contrast, review at Biodiversity Journal follows a flexible and open process featuring entirely optional anonymity and multiple types of reviewer. There, an editor appoints two or three “nominated” reviewers who must report back and several “panel” reviewers who read the paper and only comment at their discretion. Additionally, the authors may choose to open the paper to public comment during the review process.\n\nData journals all model their data validation more or less faithfully on literature peer review, but independent data validation practices and proposals are considerably more varied. On the conservative end of the spectrum, Lawrence et al. (2011) propose a set of criteria for independent data peer review44. The Planetary Data System (PDS) peer-reviews datasets through the unusual process of holding an in-person meeting with representatives of the repository, the dataset creators, and the reviewers.\n\nTwo examples from archaeology, Open Context and the Digital Archaeological Record (tDAR), illustrate the diversity of approaches to data validation. Open Context provides multiple validation processes that incorporate peer review in a way that goes beyond the simple accept/reject binary19. Each Open Context dataset is rated from one to five based not on quality per se, but on the thoroughness of the validation; a one comes with no guarantees, a three has passed a technical review, and a five has passed external peer-review. Whereas Open Context is a boutique publisher, focusing on data presentation and reuse, tDAR is a large repository primarily concerned with with collecting and preserving archaeology data for future use. tDAR is able to operate at scale by performing only technical validation and streamlining data deposition with a minimum of mandatory description. However, tDAR also serves as a platform for high-quality data publication. The repository accommodates contributors who wish to provide more information, and much of the content is deposited by digital curators who can be relied on to supply rich descriptions. Furthermore, two data paper journals, Internet Archaeology and Journal of Open Archaeological Data, recommend tDAR as a repository for their peer-reviewed data. Thus, data validation depends not only on discipline and data type, but on a host of external factors, including the goals of the organizations and researchers involved.\n\nPre-publication validation can be supplemented or replaced by post-publication feedback from successful or unsuccessful reusers. Parsons et al. (2010) suggest that “data use in its own right provides a form of review”, and go on to point out that the context of reuse demonstrates that the data is not generically “good”, but fit for some particular purpose42. The DANS repository solicits feedback from researchers who use its datasets: users are asked to rate the dataset on a one to five scale in each of six criteria (e.g., data quality, quality of the documentation, structure of the dataset)45,46.\n\n\nBeyond data publication\n\nIn a 2013 paper47, Parsons and Fox argue that thinking about data through the the metaphor of print “publication” is limiting. Diverse kinds of material are regarded as data by one research community or another and, while at least some aspects of publication apply well to at least some kinds of data, other approaches are possible. An alternative metaphor that seems to be gaining traction is “data as software”48. In some cases, it may be better to think of releasing a dataset as one would a piece of software and to regard subsequent changes as analogous to updated versions. The open-source software community has already developed many potentially relevant tools for working collaboratively, managing multiple versions, and tracking attribution. Ram (2013)49 catalogs a multitude of scientific uses for the software version control system Git, including data management. Open Context uses Git and Mantis Bug Tracker to track and correct dataset errors. Furthermore, projects such as IPython Notebook integrate data, processing, and analysis into a single package. However, scientific software struggles for recognition50 just as data does, so using it to alter or affect the academic reward system for data is a tricky prospect.\n\nUltimately, while “data as software” is promising, data is not software. Nor is it literature. The prestige and familiarity of terms like “publication” and “peer-review” are powerful, but we may have to stretch their definitions if we are determined to apply them to data.", "appendix": "Author contributions\n\n\n\nJK collected information and prepared the first draft of the manuscript. JK and CS designed the scope and direction of the study. Both authors contributed to the writing and editing of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nJK is supported by a Council on Library and Information Resources/Digital Library Foundation Postdoctoral Fellowship in Data Curation for the Sciences and Social Sciences funded by the California Digital Library and the Alfred P. Sloan Foundation.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors would like to thank colleagues at the CDL and Jodi Reeves Flores for productive discussions. Margaret Smith and Christina Doyle contributed invaluable suggestions for editing the manuscript.\n\n\nReferences\n\nCostello MJ: Motivating online publication of data. BioScience. 2009; 59(5): 418–427. Publisher Full Text\n\nSmith VS: Data publication: towards a database of everything. BMC Res Notes. 2009; 2: 113. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLawrence B, Jones C, Matthews B, et al.: Data publication. Int J Digital Curation. 2011.\n\nCallaghan S, Donegan S, Pepler S, et al.: Making data a first class scientific output: Data citation and publication by NERC’s environmental data centres. Int J Digital Curation. 2012; 7(1): 107–113. 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Eos, Transactions American Geophysical Union. 2010; 91(34): 297–298. Publisher Full Text\n\nDiederich F: Are we refereeing ourselves to death? The peer-review system at its limit. Angew Chem Int Ed Engl. 2013; 52(52): 13828–9. PubMed Abstract \| Publisher Full Text\n\nLawrence B, Jones C, Matthews B, et al.: Citation and peer review of data: Moving towards formal data publication. Int J Digital Curation. 2011; 6(2): 4–37. Publisher Full Text\n\nGrootveld M, Van Egmond J: editors. Data Reviews, peer-reviewed research data. Number 5 in DANS Studies in Digital Archiving. Data Archiving and Networked Services. 2011. Reference Source\n\nGrootveld M, Van Egmond J: Peer-reviewed open research data: Results of a pilot. Int J Digital Curation. 2012; 7(2): 81–91. Publisher Full Text\n\nParsons MA, Fox PA: Is data publication the right metaphor? Data Sci J. 2013; 12: WDS32–WDS46. Publisher Full Text\n\nSchopf JM: Treating data like software: a case for production quality data. In Proceedings of the 12th ACM/IEEE-CS joint conference on Digital Libraries, JCDL ’12, New York, NY USA, 2012; 153–156. ACM. Publisher Full Text\n\nRam K: Git can facilitate greater reproducibility and increased transparency in science. Source Code Biol Med. 2013; 8(1): 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPradal C, Varoquaux G, Langtangen HP: Publishing scientific software matters. J Comput Sci. 2013; 4(5): 311–312. Publisher Full Text" }	[ { "id": "4541", "date": "06 May 2014", "name": "Mark Parsons", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral Comments:Note: This review was written by Parsons and accepted (with some modification) by Fox. Insights likely come from conversations between Fox and Parsons, errors from Parsons.I am very glad the authors wrote this essay. It is a well-written, needed, and useful summary of the current status of “data publication” from a certain perspective. The authors, however, need to be bolder and more analytical. This is an opinion piece, yet I see little opinion. A certain view is implied by the organization of the paper and the references chosen, but they could be more explicit. The paper would be both more compelling and useful to a broad readership if the authors moved beyond providing a simple summary of the landscape and examined why there is controversy in some areas and then use the evidence they have compiled to suggest a path forward. They need to be more forthright in saying what data publication means to them, or what parts of it they do not deal with. Are they satisfied with the Lawrence et al. definition? Do they accept the critique of Parsons and Fox? What is the scope of their essay?The authors take a rather narrow view of data publication, which I think hinders their analyses. They describe three types of (digital) data publication: Data as a supplement to an article; data as the subject of a paper; and data independent of a paper. The first two types are relatively new and they represent very little of the data actually being published or released today. The last category, which is essentially an “other” category, is rich in its complexity and encompasses the vast majority of data released. I was disappointed that the examples of this type were only the most bare-bones (Zenodo and Figshare). I think a deeper examination of this third category and its complexity would help the authors better characterize the current landscape and suggest paths forward.Some questions the authors might consider: Are these really the only three models in consideration or does the publication model overstate a consensus around a certain type of data publication? Why are there different models and which approach is better for different situations? Do they have different business models or imply different social contracts? Might it also be worthy of typing “publishers” instead of “publications”? For example, do domain repositories vs. institutional repositories vs. publishers address the issues differently? Are these models sustaining models or just something to get us through the next 5-10 years while we really figure it out?I think this oversimplification inhibited some deeper analysis in other areas as well. I would like to see more examination of the validation requirement beyond the lens of peer review, and I would like a deeper examination of incentives and credit beyond citation.I thought the validation section of the paper was very relevant, but somewhat light. I like the choice of the term validation as more accurate than “quality” and it fits quite well with Callaghan’s useful distinction between technical and scientific review, but I think the authors overemphasize the peer-review style approach. The authors rightly argue that “peer-review” is where the publication metaphor leads us, but it may be a false path. They overstate some difficulties of peer-review (No-one looks at every data value? No, they use statistics, visualization, and other techniques.) while not fully considering who is responsible for what. We need a closer examination of different roles and who are appropriate validators (not necessarily conventional peers). The narrowly defined models of data publication may easily allow for a conventional peer-review process, but it is much more complex in the real-world “other” category. The authors discuss some of this in what they call “independent data validation,” but they don’t draw any conclusions.Only the simplest of research data collections are validated only by the original creators. More often there are teams working together to develop experiments, sampling protocols, algorithms, etc. There are additional teams who assess, calibrate, and revise the data as they are collected and assembled. The authors discuss some of this in their examples like the PDS and tDAR, but I wish they were more analytical and offered an opinion on the way forward. Are there emerging practices or consensus in these team-based schemes? The level of service concept illustrated by Open Context may be one such area. Would formalizing or codifying some of these processes accomplish the same as peer-review or more? What is the role of the curator or data scientist in all of this? Given the authors’s backgrounds, I was surprised this role was not emphasized more. Finally, I think it is a mistake for science review to be the main way to assess reuse value. It has been shown time and again that data end up being used effectively (and valued) in ways that original experts never envisioned or even thought valid.The discussion of data citation was good and captured the state of the art well, but again I would have liked to see some views on a way forward. Have we solved the basic problem and are now just dealing with edge cases? Is the “just-in-time identifier” the way to go? What are the implications? Will the more basic solutions work in the interim? More critically, are we overemphasizing the role of citation to provide academic credit? I was gratified that the authors referenced the Parsons and Fox paper which questions the whole data publication metaphor, but I was surprised that they only discussed the “data as software” alternative metaphor. That is a useful metaphor, but I think the ecosystem metaphor has broader acceptance. I mention this because the authors critique the software metaphor because “using it to alter or affect the academic reward system is a tricky prospect”. Yet there is little to suggest that data publication and corresponding citation alters that system either. Indeed there is little if any evidence that data publication and citation incentivize data sharing or stewardship. As Christine Borgman suggests, we need to look more closely at who we are trying to incentivize to do what. There is no reason to assume it follows the same model as research literature publication. It may be beyond the scope of this paper to fully examine incentive structures, but it at least needs to be acknowledged that building on the current model doesn’t seem to be working.Finally, what is the takeaway message from this essay? It ends rather abruptly with no summary, no suggested directions or immediate challenges to overcome, no call to action, no indications of things we should stop trying, and only brief mention of alternative perspectives. What do the authors want us to take away from this paper?Overall though, this is a timely and needed essay. It is well researched and nicely written with rich metaphor. With modifications addressing the detailed comments below and better recognizing the complexity of the current data publication landscape, this will be a worthwhile review paper. With more significant modification where the authors dig deeper into the complexities and controversies and truly grapple with their implications to suggest a way forward, this could be a very influential paper. It is possible that the definitions of “publication” and “peer-review” need not be just stretched but changed or even rejected.Detailed comments:The whole paper needs a quick copy edit. There are a few typos, missing words, and wrong verb tenses. Note the word “data” is a plural noun. E.g., Data are not software, nor are they literature. (NSICD, instead of NSIDC) Page 2, para 2: “citability is addressed by assigning a PID.” This is not true, as the authors discuss on page 4, para 4. Indeed, page 4, para 4 seems to contradict itself. Citation is more than a locator/identifier In the discussion of “Data independent of any paper” it is worth noting that there may often be linkages between these data and myriad papers. Indeed a looser concept of a data paper has existed for some time, where researchers request a citation to a paper even though it is not the data nor fully describes the data (e.g the CRU temp records) Page 4, para 1: I’m not sure it’s entirely true that published data cannot involve requesting permission. In past work with Indigenous knowledge holders, they were willing to publish summary data and then provide the details when satisfied the use was appropriate and not exploitive. I think those data were “published” as best they could be. A nit, perhaps, but it highlights that there are few if any hard and fast rules about data publication. Page 4, para 2: You may also want to mention the WDS certification effort, which is combining with the DSA via an RDA Working Group: Page 4, para 2: The joint declaration of data citation principles involved many more organizations than Force11, CODATA, and DCC. Please credit them all (maybe in a footnote). The glory of the effort was that it was truly a joint effort across many groups. There is no leader. Force11 was primarily a convener. Page 4, para 6: The deep citation approach recommended by ESIP is not to just to list variables or a range of data. It is to identify a “structural index” for the data and to use this to reference subsets. In Earth science this structural index is often space and time, but many other indices are possible--location in a gene sequence, file type, variable, bandwidth, viewing angle, etc. It is not just for “straightforward” data sets. Page 5, para 5: I take issue with the statement that few repositories provide scientific review. I can think of a couple dozen that do just off the top of my head, and I bet most domain repositories have some level of science review. The “scientists” may not always be in house, but the repository is a team facilitator. See my general comments. Page 5, para 10: The PDS system is only unusual in that it is well documented and advertised. As mentioned, this team style approach is actually fairly common Page 6, para 3: Parsons and Fox don’t just argue that the data publication metaphor is limiting. They also say it is misleading. That should be acknowledged at least, if not actively grappled with.", "responses": [ { "c_id": "815", "date": "12 May 2014", "name": "John Kratz", "role": "Author Response", "response": "Thank you for refereeing our paper and thank you especially for delivering your report so quickly.We submitted the paper as a review article, not an opinion piece, and it was reclassified somewhere along the way. I contacted an editor at F1000 about the issue, and I believe it will be switched back shortly. While there is undoubtedly a viewpoint inherent in the way we have organized the manuscript, it was our intention to deliver a timely summary of the current landscape as a foundation for future thinking, not to offer prescriptions or to endorse particular approaches. We have no shortage of opinions about data publication, and a true opinion piece may follow at some point, but our aim here was to remain fairly neutral. I think the paper you are asking for would also be valuable, but it's an entirely different paper from the one we have written.That said, your report is full of suggestions for expansion of analysis and clarification of scope that would absolutely improve the paper (e.g. the question of why some issues resist consensus more than others is an excellent one), and we will certainly address them in the next version." } ] } ]	1	https://f1000research.com/articles/3-94
https://f1000research.com/articles/3-232/v1	02 Oct 14	{ "type": "Opinion Article", "title": "Commercial antibodies and their validation", "authors": [ "JLA Voskuil" ], "abstract": "Despite an impressive growth in the business of research antibodies a general lack of trust in commercial antibodies remains in place. A variety of issues, each one potentially causing an antibody to fail, underpin the frustrations that scientists endure. Lots of money goes to waste in buying and trying one failing antibody after the other without realizing all the pitfalls that come with the product: Antibodies can get inactivated, both the biological material and the assay itself can potentially be flawed, a single antibody featuring in many different catalogues can be deemed as a set of different products, and a bad choice of antibody type, wrong dilutions, and lack of proper validation can all jeopardize the intended experiments. Antibodies endorsed by scientific research papers do not always meet the scientist’s requirements either due to flawed specifications, or due to batch-to-batch variations. Antibodies can be found with Quality Control data obtained from previous batches that no longer represent the batch on sale. In addition, one cannot assume that every antibody is fit for every application. The best chance of success is to try an antibody that already was confirmed to perform correctly in the required platform.", "keywords": [ "Based on feedback from about 10 years ago", "scepticism and mistrust towards commercial antibodies was already commonplace. Researchers in the academic environment preferred generating antibodies in-house by making use of the animal facilities in their faculties. At the time", "the availability of commercial antibodies was not as extensive as it is today", "and therefore it was unlikely that a scientist would find an antibody fitting their requirements. The present situation is quite different", "yet the complaints remain. The number of commercial antibodies has escalated in the last decade", "and so has demand. In contrast to 10 years ago when Western Blot (WB)", "ELISA and ImmunoHistoChemisty (IHC) were the most used assay types", "at present antibodies are increasingly used in more sophisticated platforms such as flow cytometry", "multiplex assays", "immune-mass spectrometry and other capture-based assays as modern technologies have made them widely accessible. Along with this increased variety of platforms", "demand for fit-for-purpose (F4P) antibodies is increasing", "while disappointment by the performance of commercial antibodies remains an ever present experience." ], "content": "Introduction\n\nBased on feedback from about 10 years ago, scepticism and mistrust towards commercial antibodies was already commonplace. Researchers in the academic environment preferred generating antibodies in-house by making use of the animal facilities in their faculties. At the time, the availability of commercial antibodies was not as extensive as it is today, and therefore it was unlikely that a scientist would find an antibody fitting their requirements. The present situation is quite different, yet the complaints remain. The number of commercial antibodies has escalated in the last decade, and so has demand. In contrast to 10 years ago when Western Blot (WB), ELISA and ImmunoHistoChemisty (IHC) were the most used assay types, at present antibodies are increasingly used in more sophisticated platforms such as flow cytometry, multiplex assays, immune-mass spectrometry and other capture-based assays as modern technologies have made them widely accessible. Along with this increased variety of platforms, demand for fit-for-purpose (F4P) antibodies is increasing, while disappointment by the performance of commercial antibodies remains an ever present experience.\n\nDespite the negativity described above, the complexity of generating F4P antibodies has made the research-antibody trade one of the fastest growing markets in the life science industry. Not only has the number of traders increased, the traders also enjoyed a substantial growth in their business. There seems to be no stop in the increasing demand for commercial antibodies for research purposes. Yet, even today, the complaints of poor performance remain the biggest problem in the research antibody industry. Attempts to release multiple antibodies targeting the same protein did not make much of a difference so far. The reasons for this are outlined below.\n\nThe scientific community is struggling with the complexity that research antibodies bring to the lab, and therefore each complicating factor is discussed separately before we can build a general picture of how to benefit optimally from commercial antibodies.\n\n\nSpecificity, affinity, background and noise\n\nThe term non-specificity is used when an antibody binds to unintended proteins. Each antibody molecule has a certain affinity to one part of the protein called an epitope, and this affinity is determined by the epitope’s amino acid sequence. It is therefore very difficult to find antibodies that react exclusively to one protein when this protein is very similar to other (closely related) proteins. Only antibodies that will bind to a unique epitope will react specifically to its intended target protein. However, most antibodies do not bind to unique epitopes and so they will cross-react.\n\nIn the case of shared epitopes between closely related proteins cross-reactivity is inevitable. Then the actual binding of the antibody may be specific, yet the antibody is deemed non-specific in relation to the intended target protein. Further diluting the antibody and optimizing blocking conditions will not work in these cases. In other words, the specificity of an antibody relies on the uniqueness of the protein part it binds to (i.e. the epitope).\n\nAn antibody specific to an epitope that is shared between one or two other (closely related) proteins may not be useless. It may still be useful in tissues or cell types where those cross-reacting proteins are not present. Or the scientist can take advantage in relating the intensities of bands representing the different proteins in Western blot.\n\nProteins unrelated to the intended target protein may have epitopes similar but not identical to the specific epitopes. Then the antibody’s affinity for the similar epitope will be lower than for the specific epitope. This will result in cross-reactivity with a proportionate lower signal, called non-specific background.\n\nNon-specific background can be reduced by further diluting the antibody. The reason is simple: by diluting the antibody only higher affinity interactions are sustained. The lower affinity interactions (to remotely similar epitopes) will not last at lower antibody concentrations. Proper blocking conditions can also help to prevent low affinity interactions. NaCl will interfere with weak hydrostatic interactions while non-polar blocking agents (for example Tween-20) will interfere with weak hydrophobic interactions between the antibodies and the unintended target proteins. Increasing the concentrations of such blocking agents may help to reduce the non-specific background.\n\nPoor experimental conditions will incur random noise and this is typically not related to the primary antibody. Lacking certain blocking components or the use of dirty containers/contaminated buffers are usually to blame. Especially in fluorescence-based assays, noise can be a big issue. There is a risk of antibodies being dismissed prematurely because non-specific background and noise are not considered and dealt wih separately.\n\nIn addition to primary antibody-derived issues, the secondary antibody can be a source of problems as well. The quality of the secondary antibody can be tested by side-by-side comparison of a complete experiment with another experiment lacking the primary antibody. Noise and background caused by the secondary antibody will become apparent in this negative control.\n\n\nAntibody types\n\nThe term “polyclonal antibodies” (pabs, as opposed to mabs for monoclonal antibodies) is ambiguous and can be the cause of some confusion. There are pabs raised against the full length protein, pabs raised against large protein fragments and pabs raised against small peptides. In addition, there are differences between antiserum, proteinA/G-purified, and antigen affinity purified pabs.\n\nThe general concept of pabs is that they represent a collection of antibodies raised against multiple epitopes and possibly against multiple proteins. When an antibody is raised to an entire protein, it is easy to see how multiple parts of the protein will generate a collection of different specificities and affinities. This is beneficial for certain applications such as immune precipitation (IP) and WB where cross-reactivity is easily spotted by the difference in molecular weight (unless there is cross-reactivity to proteins with the same molecular weight) compared to the intended target protein. Pabs can also be useful in IHC, as long as there is no cross-reaction with any other proteins present in the tissue sections of interest. The specificity of an antibody can be addressed by comparing endogenous expression levels to knock-down expression levels, comparing un-induced cells to induced cells (with elevated expression levels) or by looking at tissues where the location of the protein of interest is expected in one particular compartment or cell type.\n\nAntibodies raised against a protein fragment will show higher specificity when the amino acid sequence of the fragment is unique in the proteome. Then the above mentioned advantages of pabs are combined with the uniqueness of the antigen. Although such antibodies may not compete with the mono-specific characteristics of mabs, they can work together with mabs in sandwich type ELISA and IP; using one as the capturer and the other as the reporter.\n\nWhen the chosen antigen is a small peptide of the size of an epitope instead of a protein or a protein fragment, then the mono-epitopic characteristics of mabs are approached. It would be a prerequisite for this approach to have the peptide antibodies affinity purified using the antigen, thus giving high affinity pabs the upper hand over low affinity mabs. The cost of generating peptide-specific antibodies is also competitive with the cost of generating mabs because neither expensive screening and sub-cloning, nor antigen purification steps are required. A 10–15 amino acid peptide is easily and quickly synthesised and delivered by a specialist service. Just linking the peptide to a carrier protein and dialysis is required to have the material ready for immunization. And the peptide will subsequently be used for the affinity purification.\n\nPeptide antibodies therefore are versatile tools that combine the mono-specificity attributed to the mabs with the high affinity attributed to polyclonal antibody while keeping the production costs low. In addition, the epitope (peptide sequence) for a peptide antibody is known from the product sheet, while caution is required when a mab is used without its epitope characterized and/or published. The one advantage of mabs over peptide antibodies is the mab’s longevity. As long as the hybridoma clone remains stable, the exact same antibody will be generated. This makes monoclonal antibodies preferred over pabs for commercial kits.\n\nAntiserum and protein A/protein G purified IgG still have a mixture of affinities and specificities. From all pabs, only antigen-affinity purified antibodies will have the highest grade of specificity and affinity, and particularly so when they are peptide-derived. In my opinion peptide antibodies are ideal for research purposes, while monoclonal antibodies are ideal for long-term repeated standard assays. Yet, peptide antibodies serve as a (temporary) alternative as long as a proper mab is not available for the standard assays.\n\n\nOEM world\n\nThe vast majority of vendors do not manufacture all the antibodies on the catalogue, and most of their antibodies have been obtained from a wide variety of different manufacturers from all over the world under OEM agreement (Other External Manufacturer). Such an agreement usually has a clause to forbid the supplier from publishing which of their products are sold by their OEM vendor. The vendors keep up the appearance that they themselves are the primary source of all their antibodies. This enables them to keep QC data on the product sheet that were generated many years ago thus keeping the sales going, while the actual antibody that generated these data may have sold out and has been replaced by successive other batches (from different animals) and the current batch on sale may no longer be able to generate such data at all.\n\nEven monoclonal antibodies suffer from batch-to-batch variations, but not to such severe extent as some types (see above) of polyclonal antibodies. Nonetheless, certain hybridoma clone numbers are still being used for decades while, just like with cell lines, hybridomas cannot be the same after so many passages anymore. It is therefore misleading to use QC data that were generated decades ago, unless the current batch has proven to still be capable of generating such data (in which case one might as well show the latest version of the data).\n\nAssay developers are advised to buy antibodies straight from the manufacturer and ask for a free validation sample from a large batch in stock. Once validated for the required platform, the same batch then can be purchased in bulk so to prevent batch-to-batch variations during the entire project. Identifying the manufacturer can be a challenge though, and the only way to find them is to start looking for overlap between the product sheets from the different vendors. This way, a list can be generated at which point one can guess who the manufacturer was based on additional details still present on the manufacturer’s product sheet but not elsewhere.\n\nVendors accrue data from their own customers or from their own QC department, thus making the OEM product look unique. This way the same antibody can show different QC data on different catalogues. And while batches run out and are being replaced by others, it can happen that a vendor has still some of the old batch in stock, while another vendor will keep the QC data obtained from the former batch on their product sheet. From this moment on customers start to buy products that are no longer necessarily reflected by their product sheet.\n\nVendors do not only obtain their antibodies from the original manufacturers. There is a network of vendors obtaining each other’s catalogue items. Consequently, the same antibody starts to occur several times in one catalogue: one time with the current QC data provided by the original manufacturer, and one or more times with QC data obtained from the other vendor’s direct customers or QC department. Potentially, assay developers buy several antibodies from several vendors thinking they are buying different antibodies, yet a number of them originate from the same manufacturer’s catalogue number.\n\n\nReasons of performance failures\n\nAntibodies deteriorate by repeated freeze/thaw cycles. Such cycles should be kept to an absolute minimum. One way is to keep aliquots frozen at all times and have one aliquot in the fridge for daily use until finished. Primary antibodies usually come with preservatives to keep them good at 4°C for many months. Please be aware that some freezers are opened extremely often during a working day and then antibodies stored at the front may endure freeze/thaw cycles because of the frequent and long browsing and possibly in combination with the summer sun shining straight in. When this applies, antibodies are best kept in a different freezer that is less often opened (eg. -80°C).\n\nWestern blot is seen by many as the easiest and most straightforward type of immune assay. This systematic underestimation is thought to be holding back science at a great scale. With so many pitfalls unrecognized, many good antibodies are dismissed after one or two poor experiments. The Western blot is possibly the most complicated immune assay because of the many layers where the assay can fail. The most trivial problems and their remedies are highlighted in Table 1.\n\nPotential problems may have already occurred during preparations of the biological material to be analyzed, well before the assay takes place. Proteolysis and oxidation are two notorious factors that will introduce low reproducibility of results when comparing different lysates from the exact same cell type with each other. The conditions (including temperature) of protein separation in SDS PAGE will influence the banding patterns. And finally, different tissue types and cell types do not necessarily give mutually identical patterns in WB. It depends on the type of protein of interest and whether any posttranslational modifications (PTM) and/or degradation may take place in a different fashion in each cell type. The PTMs may be prone to (partial) removal during the lysate preparation by endogenous enzymes. Proper inhibitors (making sure they did not get inactivated during preparation) should be added to the lysates to minimize such impairments. Hence, a commercial antibody shown to work properly in one tissue type or species should still be validated in other tissue types or species. And it is best advised to generate several lysates of the same cell type or tissue type at different days before starting analysis of all of them, side by side.\n\nWhen cells, tissue blocks or their sections have been stored after dehydration, the structure of the proteins in the cells will have changed thus affecting the binding of antibodies. Antigen/epitope retrieval will restore this, but its success is very dependent on the method of retrieval and the method may have to be adjusted from antibody to antibody (or from target protein to target protein). Heat induced epitope retrieval (HIER) can be done at pH6 and at pH9 by either high pressure steaming or by microwave. The quality of results can heavily depend on the conditions (pH value, temp, the heating duration, and the method of heating). When all attempts through HIER fail, one could opt for protease induced epitope retrieval (PIER), although some scientists prefer PIER over HIER as their sole approach.\n\nWhen fresh tissues have been fixed in alcohols or acetone, one usually does not store the samples, and cryosectioning and subsequent probing with the antibodies go ahead in one flow. In this case epitope retrieval may not be required. I would however recommend epitope retrieval if the tissues have been stored long-term in the alcohol or acetone before cryosectioning. The dehydration of the tissues may mimic the paraffin embedding procedure, particularly in the case of storage in the hydrophobic acetone. Hence, rehydration of the tissue during the removal of the alcohol/acetone may still not be enough to expose the epitopes fully (again) after long term dehydrated storage.\n\nFluorescent labels are commonly used in Flow Cytometry (FC), ImmunoCytoChemistry (ICC) and in IHC as well. The major issue with fluorescence is the noise. Because of the high sensitivity of this detection type, noise is much more prevalent compared to other less sensitive detection types.\n\nAs with all assays, different dilutions of the primary (including one without primary) will have to be compared in order to appreciate the level of constant noise. One has to be careful in choosing the right blocking agents, but more importantly, one has to take into account the presence of endogenous molecules (even at low abundance) that bind to either the fluorophore itself or to its conjugated carrier (for example avoid using streptavidin-fluorophore when there is endogenous biotin, and avoid using anti-IgG-fluorophore when there are endogenous Ig-receptors).\n\nSome antibodies will not pull their target proteins down under certain conditions. Particularly when working with linear epitope antibodies (e.g. peptide antibodies) one is advised to compare native conditions with denatured conditions (reduced and in presence of 0.1% SDS) as a positive control.\n\nDuring the classic method of IP, a network of antigen and polyclonal antibodies is precipitated and analyzed. A polyclonal antibody generated to the entire protein is essential for this method as many epitopes are involved in the network formation that is precipitated. However, when the target protein is bound to one or more other proteins, the variety of epitopes for the antibody to bind to become limited and the network may not be stable enough for this classic approach. It is therefore recommended to have a control of denatured protein IP side-by-side with the actual experiment.\n\nWhen using epitope-specific antibodies (monoclonal or peptide-polyclonal) the target protein (complex) is brought down by beads. This approach makes the scientist independent on an antibody-antigen network to form. When the right epitope-specific antibodies are used, extra information can be generated about the binding sites for the interacting other proteins.\n\nThis assay type represents any micro-well formatted immunoassay. They all have in common that one reagent is coated to a stationary phase (usually the bottom of the well), and other reagents react with the coated reagent proportionally to the content of the analysed material within a natural matrix. Matrix could be a body fluid (plasma, serum, urine, etc), a culture supernatant, or a buffered solution spiked with biological material from which one constituent needs quantifying.\n\nThe biggest hurdle in such assays is the notorious matrix effect. To put it simple: the matrix contains molecules that interfere with the reaction between the antibodies and the antigen to be quantified. This interference can be visualized by making serial dilutions of the matrix. When the matrix is diluted with a factor two and the measured antigen therein does not read a reduction by a factor two along the way, you have established matrix effect. Also when readings do not correlate to the levels of antigen spiked into the matrix of interest, there will be matrix effects. Matrix effects are best dealt with by diluting the matrix in assay buffer to such extent that the antigen can be quantified without interference from the matrix. It is essential to establish matrix effect every time a new assay is set up. When a new antibody is introduced in an already existing assay, the matrix effect may respond differently from the previous antibody. A calibration curve needs to be made in the same matrix and run in parallel with each assay.\n\nPolyclonal antibodies may perform better after being pre-adsorbed to abundant serum proteins as a blocking step. This should be a compulsory step when using the antibody in matrix containing high levels of serum protein. One needs to be aware that most commercial primary antibodies have not been pre-adsorbed to serum proteins. Some detergents may also minimize matrix effect.\n\nOne should not jump to conclusions after a single experiment. Even positive data may sometimes be false positive and lack of signals may be due to trivial factors that can be solved by systematic trouble shooting. Therefore conclusions can only be drawn once identical results have been obtained by the same experiment carried out at different times.\n\nAnyone working in a subtropical climate (eg. southern state of the USA) knows how hot it becomes on a sun baked parking space during summer and how temperatures can soar inside a delivery van parked out there while taking care of a delivery. Antibodies are inherently made to work at 37–42°C, but it remains a protein and it can cook until inactive when temperatures go well over 50°C. Every antibody is different from the next and the majority will survive the above mentioned conditions. When antibodies in solution arrive on frozen icepack, one can assume that the integrity of the antibody has been maintained during transport. Lyophilized antibodies are more resistant. It is the user’s responsibility to ensure that the ordered antibody is received in proper packaging (particularly during hot weather conditions) and complain to the vendor when this is not the case. When an antibody does not perform as expected based on the product sheet and the antibody was delivered under hot conditions, it is time to ask for replacement.\n\nToday’s advanced internet facilities enable vendors to search for publications describing successful use of their antibodies. However, some publications mistakenly attribute an antibody to the wrong vendor. And sometimes an antibody is correctly reported but no data generated by it are to be seen or presented in the paper, nor in the supporting documentations. In such cases the internet search delivers false-positives, and examples can be found in each vendor’s catalogue. Many such papers are inaccessible to the vendors and therefore they cannot always double check themselves. Although this is a relatively rare phenomenon, it is real all the same and therefore the customer is advised to double check the contents of the referenced paper when such endorsement is a requirement for the scientist to purchase this antibody. Each vendor will appreciate the feedback when a customer identifies a false-positive reference, and we urge customers to contact the vendor when such reference has been identified so they can remove it from the product data sheet.\n\nAlthough there is a recent change in trend, the vast majority of publishers still do not demand to specify the used antibodies by their catalogue number in scientific papers. Since most catalogues have more than one antibody to one protein, a mere description of an antibody from vendor X to protein Y is not sufficient and prevents peers from replicating the described experiments.\n\n\nGeneral principles\n\nEvery researcher in the lab will have different wishes and demands on how an antibody should perform. Ideally, an antibody meets all demands one can think of. Unfortunately this does not always happen. Each antibody has its own unique characteristics. It may work very well in one or two types of assays (for example in WB and IHC), but not in other platforms. If one needs an antibody for quantification in micro-well format, one should not test in WB or IHC. Many WB antibodies do not work in IHC and many IHC antibodies do not work in WB. Yet, all combinations of the above are feasible, and to certain target proteins all antibodies work in all applications tested.\n\nSadly, there are also many proteins out there that refuse to generate antibodies fit for any application. Hence, the choice of target protein is a big factor determining the versatility of antibodies or whether it is fit for purpose at all. The host species can also be a factor. It is commonly known that when mammals fail to produce a decent antibody one has to generate bird antibodies (chicken).\n\nValidation can also be restricted by a regulatory environment. Laboratories regulated by Good Laboratory Practice (GLP) will have to follow fixed procedures for antibody validation (see http://www.mhra.gov.uk/Howweregulate/Medicines/Inspectionandstandards/GoodLaboratoryPractice/Structure/ and http://www.epa.gov/compliance/monitoring/programs/fifra/glpsops.html). In this case, the quality control data on the product sheet are hardly relevant. The operator in the GLP lab will have to follow the obligatory procedures from the start anyway. Here, one has to decide on purchasing the antibody with the highest chances of success. Here the decision can go wrong when one picks an antibody with superior QC data that is from irrelevant applications. One tends to think that an antibody without QC data is less likely to be successful for the required assay in the GLP lab than an antibody with nice WB and/or IHC data. It is a difficult choice to make as each purchase has to undergo this obligatory and therefore expensive validation procedure. The best choice is an antibody that already has proven itself in the relevant assay type. It is therefore recommended to first try a panel of different antibodies (from different manufacturers) in a much cheaper feasibility study before making that choice. It is worth asking manufacturers for antibodies that cannot be found on any catalogue. Many antibodies are waiting to be tested in assays not accessible to the manufacturers, and so they are not for sale (not working in WB or IHC) and a free sample is often made available if feedback on the results is promised in return.\n\n\nHow to validate an antibody\n\nAn antibody is meant to bind to the protein intended. This confirmation is a minimal requirement for the product’s datasheet. One should never purchase a product without this confirmation on the product sheet. Usually this confirmation is established by direct ELISA with a titre. Alternatively, a recombinant protein or purified protein (or fragment thereof) may be stained in WB. This is not yet a validation of any kind! It merely confirms that the antibody has a certain affinity to the intended target protein.\n\nValidation starts with comparing the antibody’s affinity to the intended target protein with its affinity to all other proteins occurring in the natural environment of the intended target protein. In other words, the antibody needs to be able to specifically bind to its intended target while it does not bind to the vast majority of all other molecules that naturally surrounds the target. For this reason it is good practice to compare the binding of the antibody on two identical mixtures of proteins, one with the target and one without the target. This may translate into comparing matrix, lysate or tissue containing endogenous target levels with matrix, lysate or tissue containing knocked-down or knocked-out target levels. Or as an alternative, matrix, lysate or tissue with low target levels compared to matrix, lysate or tissue with artificially increased target levels.\n\nMore often than one would wish, the results of the above mentioned tests are not going to be black and white. When successful, a clear preference is observed to the intended target, but with a certain level of background. At this stage one has to optimize conditions so to increase the signal/background to acceptable levels. One has to take into account that when the antibody is binding to common epitopes, this antibody is going to be cross-reactive with related proteins sharing these epitopes when also present in the mix. This cross-reactivity then invalidates the antibody and a better antibody needs to be identified. When the antibody is mono-specific to one defined and unique epitope, one has to take into account that such an antibody will still bind to similar epitopes albeit at lower affinity. The background occurring from proteins with such epitopes can be reduced by further diluting the antibody and by reducing the primary incubation time. Consequently negative control tissues or cells still show signals when the antibody was used at too high concentration. Please note that this principle is hardly recognized by the world of antibody therapeutics, thus posing a risk that a therapeutic antibody at high dose will bind to other proteins!\n\nBackground can also derive from added reagents required for signal reporting. A bad secondary antibody can be identified by comparing the complete assay with the same assay but without the primary antibody. Finally, random noise is most likely produced by the reporting chemicals when the blocking conditions and or buffer constituents have not been optimal.\n\nIn WB, background can be seen as extra bands, while noise can be seen as random spots. In IHC, background will still stain certain structures in the cell (albeit different from the expected structures), while noise will show stains overlapping different cellular structures, thus showing lack of specificity of the stain itself. In fluorescence, noise will become apparent as a constant when different dilutions of the primary are compared.\n\nThe complexity of commercial antibodies is for a great deal owed by the OEM agreements. Everyone should be aware that the same antibody appears multi-fold on many catalogues worldwide and not always with identical QC data. Another complication is the formulation and antibody type on offer. A monoclonal antibody can be offered as a purified IgG in known mg/ml concentration, while another catalogue offers the same monoclonal antibody in culture media without any specifications. A polyclonal antibody can be offered as affinity purified by one catalogue and as antiserum by another. Scientists should be aware that when the (exact) epitope is not given or known, the antibody needs a more robust validation study than when the epitope is specified.\n\nThe many layers of complexity outlined above give rise to combinations of problems that are challenging to solve. Only systematically going through each layer separately, one can finally validate an antibody without running the risk of dismissing a precious product.\n\n\nConcluding remarks\n\nRecently, new initiatives are being developed to aid the scientists with their validation efforts; F1000Research is launching a permanent article collection to provide a platform for scientists to publish their antibody validation studies. In addition, publishers are starting to ask the authors to report the catalogue number of each antibody used in their publications. Also, last February the Resource Identification Initiative was launched (http://scicrunch.com/resources) which provides permanent identifiers for lab resources such as antibodies, model organisms and software tools and aims to make these tools universally identifiable. These are significant steps towards our goal of increasing the confidence the scientific community has in commercial antibodies.", "appendix": "Competing interests\n\n\n\nThe author is the Chief Scientific officer of Everest Biotech Ltd and has written this manuscript without the intention of jeopardizing anyone’s business. In fact Everest Biotech as a manufacturer benefits from any increased business that all antibody vendors enjoy as a result of the growing trust in commercial antibodies. It is perceived that increased confidence in commercial antibodies will increase the trade for all vendors and manufacturers.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in the funding of this work." }	[ { "id": "6362", "date": "07 Oct 2014", "name": "Fridtjof Lund-Johansen", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWhat does the antibody manufacturer say? We all know what scientists say about commercial antibodies, but what does the antibody manufacturer say? In this opinion article the CEO of an antibody manufacturing company points out that many problems may be solved if the customers pay attention to common pitfalls in antibody applications and understand how the reagents are manufactured and marketed. The author begins with a general description of terms such as specificity, cross-reactivity, and background and continues with explaining differences between monoclonal and polyclonal antibodies. Even experienced users need to be reminded that polyclonal antibodies do not necessarily detect multiple epitopes, that there is extensive lot to lot variation in performance and that purified IgG is not the same as affinity purified antibody. Those who use assays such as western blotting, immuno-histochemistry and ELISA on a regular basis will most likely recognize the pitfalls that are described. Experts can surely produce far longer and more detailed lists with suggestions for troubleshooting. However, for the much larger number who use these assays only now and then, the article should serve as a useful reference. The author flags that he is CEO of a manufacturer that specializes in the production of anti-peptide polyclonal antibodies. Thus, one may expect that he has strong opinions about the relative performance of antibodies raised against peptides or full length proteins, respectively. One may of course agree that an antibody that has been raised and affinity purified against a peptide can be said to recognize a single epitope. Yet, there is no reference to any studies where anti-peptide antibodies have been shown to be generally more specific than reagents raised against full length proteins. For the more experienced antibody users, the open and honest description of the antibody industry is likely to be the most interesting part of the article. Most users are probably not aware of the extensive practice of buying and selling products that occurs between suppliers. It is also very useful to be warned that the validation data in product specification sheets are often not generated by the seller of the reagent and that they may not be representative of the particular lot that is sold at any given time point. The fact that the same reagent is sold under different names and with different validation data is another very interesting detail. This is an opinion article, and should be read as such. This reviewer tends to disagree at several points. Yet, hearing what the antibody manufacturer says is well invested time.I would recommend the following changes: The matrix effect: page 5 right, first paragraph. The sentence \"When the matrix is diluted with a factor two and the measured antigen does not read a reduction by a factor two along the way...... \" may be misinterpreted. If the sample is diluted and the signal remains the same, the antigen may be saturating. I was not aware that antibodies may perform better in ELISA for low abundance serum proteins if they are adsorbed against human serum proteins first. I do not understand why this would be the case, and a reference would be good. I would remove the discussion about therapeutic antibodies on page 6. Therapeutic antibodies undergo testing far beyond that of antibodies used for research purposes. I would not call extra bands in a WB \"background\" but rather cross-reactivity.", "responses": [ { "c_id": "1022", "date": "07 Oct 2014", "name": "Jan Voskuil", "role": "Author Response", "response": "Many thanks for your well formulated feedback. I will systematically respond to the points you are raising:The paper is not meant to only give an idea what the scientist in the lab should do. It should be clear that certain responsibilities also lie with the vendors. This paper should not be split into two sections: one to tell the users what to do and one to tell the users how the industry works. This paper is meant to give an unbiased overview of how trust in commercial antibodies can be gained from the market. In my opinion an integrative approach and total transparency can achieve this.I am not in agreement that this paper is merely fit to advice scientists who only now and then uses a particular assay and who is in need of technical support. Based on a decade of feedback from both industry and academics I can assure you that all experts need some reminding from time to time. I am convinced that my explanation on how peptide antibodies differ from other polyclonal antibodies will be well received.I hope I am convincing enough to make a case for antibodies raised and affinity purified using a short peptide approaches the mono-specificity of monoclonal antibodies, while antibodies generated to larger protein fragments or to entire proteins would per definition contain a mixture of antibodies to many different part of the antigen. This logic does not require evidence by reference.By the way, I am CSO, not CEO of the company. I bear responsibility for the quality and scientific content on the catalog and web pages, while the CEO (another person in this case) bears end-responsibility for the entire enterprise.I appreciate your recommendations. Once all reviews are in, I will pay a little more attention to the passages that you mention. I do have to maintain though, that extra bands in WB are considered background when you can fade them out upon further dilution of the primary antibody, while this is not the case with cross-reaction (to proteins with identical epitopes). This matter highlights that even experts sometimes need a reminder." } ] }, { "id": "6304", "date": "10 Oct 2014", "name": "Simon Glerup", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nApart from research funding, difficulties in finding good research antibodies is probably one of most limiting and disabling factors in life science research.The opinion article by Dr. Voskuil contributes with important insight into the research antibody industry. In particular, I think that most scientists are unaware of the reselling and relabeling of commercial antibodies that apparently is common practice in the industry. The article also meticulously puts forward a set of guidelines for validating the use of research antibodies, which are of broad interest to the scientific community.It was a pleasure to read and I recommend it for indexation.", "responses": [] }, { "id": "6302", "date": "10 Oct 2014", "name": "Andrew D. Chalmers", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis opinion article gives Dr Voskuil’s view on commercial antibodies and how researchers can make best use of them and potentially avoid a lot of frustration at the bench. He bases his opinions on his extensive experience of commercial antibodies gained during his time as Chief Scientific Officer of Everest Biotech.The article is wide ranging and gives many useful pieces of advice that I hope will help researchers and prompt some interesting discussion. I was pleased to see the fact that many suppliers will offer the same antibody (OEM) and the fact that properly citing antibodies is important, were both covered.One area that could have been covered in more detail is that the three dimensional structure of the epitope is important, both when the antibody is raised and in the applications that are being carried out, for example denaturing conditions in western blotting and native folding in IPs. However, no article can cover everything and this one is already wide ranging, so I don’t think it is an essential correction. There were a few small issues that could be corrected/changed if a revised version is produced.I would have thought tween-20 should be referred to as a detergent rather than non-polar? In table 1, “reduce the lysate” this could be clearer, does it mean add the reducing agent just before loading or reducing the amount of lysate? page 5, “to put it simple” should read “to put it simply” Given this is an opinion article, it might be good to have a box summarizing Dr Voskuil’s experience (which is extensive) to explain to readers why he is qualified to provide the commentary.", "responses": [ { "c_id": "1024", "date": "10 Oct 2014", "name": "Jan Voskuil", "role": "Author Response", "response": "Many thanks for your kind and helpful comments. I will certainly look at submitting a version 2. I concur that a summary of my experience may add value, and will consider adding this to the revised article. In the meantime my experience can also be found in my LinkedIn profile. Your recommendations are highly appreciated. However, Tween-20 is a non-ionic detergent, but not all detergents are non-ionic: SDS and urea for example are ionic detergents (also known as chaotropic agents). Sorry that I have left out the three-dimensional epitopes. I did it on purpose since I think there is another opinion paper to be written on this very (in my opinion yet unsettled) subject. Besides, it does not add to the objective of this paper: Giving the reader insight into the pitfalls of commercial antibodies and how to avoid wasting unnecessary resources." } ] } ]	1	https://f1000research.com/articles/3-232
https://f1000research.com/articles/3-189/v1	12 Aug 14	{ "type": "Research Note", "title": "Observations on spiny dogfish (Squalus acanthias) captured in late spring in a North Carolina estuary", "authors": [ "Charles Bangley", "Roger Rulifson", "Roger Rulifson" ], "abstract": "Five spiny dogfish were captured in early-mid May during gillnet and longline sampling targeting juvenile coastal sharks in inshore North Carolina waters. Dogfish captures were made within Back Sound and Core Sound, North Carolina. All dogfish were females over the size at maturity, and were caught at stations 1.77-2.74 m in depth, with temperatures 22.9-24.2 °C, 32.8-33.4 ppt salinity, and 6.9-8.0 mg/L dissolved oxygen. Stations where dogfish were captured were approximately 6.5-15.7 km from the nearest inlet and 43.4-247.1 m from the nearest seagrass bed. These observations are among the latest in the spring for spiny dogfish in the southeastern U.S. and occurred at higher temperatures than previously recorded for this species. It is unclear whether late-occurring spiny dogfish in this area represent a cryptic late-migrating or resident segment of the Northwest Atlantic population.", "keywords": [ "The spiny dogfish (Squalus acanthias) is a small", "highly migratory coastal shark common in Northwest Atlantic waters from Newfoundland to Cape Hatteras (Stehlik", "2007). After signs of population disturbance resulting from overfishing", "stringent fishery management regulations were put in place for this species", "and the population was considered recovered within ten years of implementing a fishery management plan (Rago & Sosebee", "2010). Such a swift recovery was unexpected for this species due to its life history characteristics: spiny dogfish in the Northwest Atlantic are not reproductively mature until an age of 12 years", "have a 2-year gestation period", "and give birth to only 1–15 young (Nammack et al.", "1985)." ], "content": "Introduction\n\nThe spiny dogfish (Squalus acanthias) is a small, highly migratory coastal shark common in Northwest Atlantic waters from Newfoundland to Cape Hatteras (Stehlik, 2007). After signs of population disturbance resulting from overfishing, stringent fishery management regulations were put in place for this species, and the population was considered recovered within ten years of implementing a fishery management plan (Rago & Sosebee, 2010). Such a swift recovery was unexpected for this species due to its life history characteristics: spiny dogfish in the Northwest Atlantic are not reproductively mature until an age of 12 years, have a 2-year gestation period, and give birth to only 1–15 young (Nammack et al., 1985).\n\nA possible hypothesis for swifter than expected recovery is that currently cryptic migratory behavior moves some portions of the spiny dogfish population out of range of both fishing pressure and fishery-independent surveys used to assess spiny dogfish stocks. According to data from the National Marine Fisheries Service (NMFS) trawl survey, spiny dogfish exhibit a general north-south migration pattern along the U.S. Atlantic coast, occurring in North Carolina and Virginia waters south to Cape Hatteras during the winter and spring, and moving north to the Gulf of Maine and Canadian waters in the summer and fall (Stehlik, 2007). However, spiny dogfish movements and distribution may not conform to this pattern. Mark-recapture studies based in both the U.S. and Canada provide evidence for more complex migratory behavior, with little migratory overlap between the Gulf of Maine and Atlantic waters south of Cape Cod and inshore-offshore migrations among dogfish remaining in Canadian waters year-round (Campana et al., 2007). More recently, spiny dogfish tagged with pop-up satellite tags in the Gulf of Maine were tracked moving off the continental shelf, providing more evidence for inshore-offshore migrations (Sulikowski et al., 2010).\n\nSpiny dogfish also occur south of Cape Hatteras, with large aggregations encountered during the winter and early spring from Cape Lookout to Cape Fear in North Carolina waters (Rulifson & Moore, 2009) and along the South Carolina coast (Ulrich et al., 2007). Spiny dogfish south of Cape Hatteras tend to occur in shallower water closer to shore than conspecifics north of Cape Hatteras (Rulifson & Moore, 2009, Rulifson et al., 2012). Acoustic telemetry data suggest that these sharks are part of the population that migrates between Cape Hatteras and Cape Cod (Rulifson et al., 2012), and seem to occupy southern waters between November and April (Ulrich et al., 2007, Rulifson et al., 2012). Despite this consistent behavior among acoustically tagged sharks, Rulifson et al., (2012) captured several spiny dogfish by hook and line at Cape Lookout on June 1, 2010, long after the end of the overwintering period for this species. Here we report further observations of spiny dogfish occurring in southern waters long after their expected migration north.\n\n\nMethods\n\nSpiny dogfish were captured during a survey designed to assess habitat selection by juvenile coastal sharks in North Carolina inshore waters. The sampling area encompassed the entirety of Back Sound from Beaufort Inlet to Cape Lookout, and extended north through the southern extent of Core Sound into Jarrett Bay (Figure 1). Sampling also occurred within Newport River from Beaufort Inlet to the Newport Marshes. Sampling locations were chosen with the goal of sampling three different habitat types; seagrass beds, shallow sand flats, and deep channels.\n\nSharks were captured using bottom-set longline and gillnet gear. Longline gear consisted of a 274.32 m mainline 6.35 mm in diameter with 50 gangions comprised of a longline clip with a swivel, a 1 m leader of 136.08 kg test monofilament line, and a size 12/0 circle hook, attached at 5–7 m intervals. Gillnet gear measured 50 m in length and 2.4 m in height, and was comprised of eight panel sections of monofilament mesh measuring 7.5, 10, 12.3, 15.5, 17.1, 21, 25.6, and 31 cm stretched, respectively. Both gears were soaked for 30–60 minutes. Where space allowed, both gears were deployed within 100 m of each other and allowed to soak simultaneously; otherwise only one of the gear types was deployed. At each sampling location, depth (m) was recorded using an onboard depth sounder, and temperature (°C), salinity (ppt), and dissolved oxygen (mg/L) were measured using a YSI model 85. Distance from the nearest inlet and distance from the nearest mapped seagrass bed were calculated by plotting the sampling locations in ArcGIS 10.1 and measuring the straight-line distance (m) between the sampling stations and those geographic features. Mapped seagrass locations were taken from ArcGIS shapefiles of submerged aquatic vegetation generated by the Albemarle-Pamlico National Estuary Partnership (APNEP, 2008).\n\nAll captured sharks were identified to species and sex, fork length (FL, mm), and total length (TL, mm) were recorded. Signs of life-history stage such as umbilical scarring and visible pregnancy were also recorded. All batioids were identified, and sex and disc width (DW, mm) were recorded for each individual. All other bycatch organisms were identified, counted, and released.\n\n\nResults\n\nA total of 52 stations were sampled from March 21 to July 1, 2014, 12 of which were sampled using longline gear and 31 of which were sampled by gillnet. Sampling encompassed Newport River, the western half of Back Sound through Middle Marsh, and Core Sound between Cape Lookout and Jarrett Bay (Figure 1).\n\nSpiny dogfish were captured in May during two gillnet sets (Figure 2, Data Set 1). The first capture event took place on May 6 during a gillnet set deployed at the northeast corner of Middle Marsh. The gear was deployed at 1325 hours and allowed to soak for 30 minutes. Four adult female spiny dogfish ranging from 849–905 mm TL were captured. The site of capture was 2.74 m in depth, with a temperature of 22.9°C, salinity of 32.8 ppt, and 8.0 mg/L dissolved oxygen. A bluntnose stingray (Dasyatis say, DW = 450 mm) and a bullnose ray (Myliobatis freminvillii, DW = 458 mm) were also captured in this set. Spatial analysis showed that this site was 6526.40 m from the nearest inlet and 43.43 m from the nearest mapped seagrass area.\n\nThe second capture event occurred on May 18 on the north side of Davis Island in Jarrett Bay. Time of gillnet deployment was 1442 hours and soak time was limited to 30 minutes. One spiny dogfish was snared in the mesh by its dorsal spines but was able to break free and escape before it could be brought aboard. Visual estimate placed the TL of this shark within the range of those captured earlier (850–900 mm). A depth of 1.77 m, temperature of 24.2°C, salinity of 33.4 ppt, and dissolved oxygen of 6.88 mg/L were recorded at this site. Other species captured at this site included one cownose ray (Rhinoptera bonasus, DW = 414 mm), one bullnose ray (DW = 458 mm), and four harvestfish (Peprilus alepidotus). This site was 15670.63 m from the nearest inlet and 247.08 m from the nearest mapped seagrass bed.\n\n\nDiscussion\n\nThese observations represent the highest reported temperatures and latest occurrence for spiny dogfish in their overwintering habitat off the Southeastern U.S, with the exception of those captured on June 1, 2010 by Rulifson et al., (2012). The presence of these sharks in North Carolina waters in late May at temperatures above 22°C is inconsistent with current information on spiny dogfish distribution and environmental preferences. Whether these observations point to unique behavior among spiny dogfish occurring near Cape Lookout or the limitations of other sampling efforts for this species is uncertain.\n\nSpiny dogfish have been consistently observed overwintering south of Cape Lookout. Bearden, (1965) reported that spiny dogfish were captured in trawl surveys within South Carolina waters as far south as Port Royal Sound between December and March at water temperatures ranging 7.5–12.0°C. Year-round gillnet and longline sampling along the South Carolina coast only captured spiny dogfish at temperatures below 14°C between January and March (Ulrich et al., 2007). In the vicinity of Cape Fear, North Carolina, Thorpe & Beresoff, (2000) captured spiny dogfish in commercial gillnet gear from December-April, though the sharks were most abundant in February and March and at temperatures less than 13.9°C. In contrast, only one spiny dogfish was captured during gillnet sampling from May-September in the same area (Thorpe et al., 2004). Schwartz, (2003) reported that spiny dogfish could occasionally be encountered along the coast of the Carolinas until May, but temperatures higher than 18°C triggered migration offshore and northward.\n\nSeasonal habitat preferences inferred from trawl survey and mark/recapture studies focused on the area between Cape Hatteras and the Scotian Shelf are consistent with observations from areas further south. Spiny dogfish occurring between Cape Hatteras and the Gulf of Maine mostly occurred in North Carolina waters during winter and spring, and were distributed between New England and Canadian waters during summer and autumn (Campana et al., 2007, Stehlik, 2007). Within this area, spiny dogfish were captured primarily in the 5–17°C temperature range (Sagarese et al., 2014). Spiny dogfish occurred at temperatures up to 20°C in Massachusetts inshore waters in autumn, but were most abundant within the 6–15°C range (Stehlik, 2007). Acoustically-tagged spiny dogfish were only detected near the Hatteras Bight between mid-December and early April, with the majority of detections occurring in February and March, and appeared to make inshore-offshore movements in search of cooler temperatures (Rulifson et al., 2012).\n\nThough the observed presence of spiny dogfish was inconsistent with previously documented environmental preferences, observations were consistent with other aspects of spiny dogfish behavior. All of the measured sharks were female and well within size at maturity for this species (799 mm TL, Nammack et al., 1985). This is consistent with observations from nearshore South Carolina waters, where 91.9% of spiny dogfish captured during shark surveys were females, and 80% of females were mature (Ulrich et al., 2007). Size is inversely correlated with depth in spiny dogfish, with the largest individuals occurring in shallow, nearshore waters (Methratta & Link, 2007), and mature females occur at significantly higher temperatures and lower depths than other demographic groups (Sagarese et al., 2014). Dell’Apa et al., (2014) observed a greater proportion of females among spiny dogfish captured by gillnet and longline in Massachusetts Bay, an area with a gradually sloping depth profile, than along the eastern shore of Cape Cod, where depth drops rapidly near shore. Spiny dogfish feed primarily on schooling pelagic fishes (Link et al., 2002), and the harvestfish co-occurring with them in Core Sound may represent a potential food source within this estuary.\n\nLittle is currently known about spiny dogfish habits within estuarine waters. Spiny dogfish observed during this survey penetrated relatively far into the estuary (6–15 km from the nearest inlet) and were captured close to seagrass habitat areas. Sharks can exert top-down influences that can have far-reaching direct and indirect effects on the ecology of estuarine environments (Heithaus et al., 2012). Determining whether spiny dogfish are ecologically important within North Carolina inshore waters will require further observation.\n\nIt is unclear whether these late-occurring spiny dogfish represent a fluke occurrence or previously unrecognized behavior. Spiny dogfish remaining within North Carolina waters into May and June have also been reported by Rulifson et al., (2012) and Schwartz, (2003), but have not been documented by most studies. The NMFS seasonal trawl surveys only sample North Carolina waters during the early spring and autumn and may not account for spiny dogfish occurring in the area at other times of the year, but migration out of southern waters in spring has also been suggested by gillnet and longline surveys capable of capturing sharks year-round (Thorpe & Beresoff, 2000, Thorpe et al., 2004, Ulrich et al., 2007), as well as acoustic telemetry (Rulifson et al., 2012). Our observations also represent the highest temperatures reported for this species in the southeastern U.S., suggesting that the thermal range for this species may be wider than previously estimated.\n\nPrevious studies have shown that spiny dogfish migration and habitat use patterns may be more complex than previously thought (Campana et al., 2007, Rulifson & Moore, 2009, Sulikowski et al., 2010). The presence of a late-migrating or resident population segment near Cape Lookout may have important implications for spiny dogfish fishery management. Future year-round surveys conducted in tandem with telemetry studies focused on late-season individuals may help explain the unusual spiny dogfish behavior in this area.\n\n\nData availability\n\nF1000Research: Dataset 1. Date, time, and location of spiny dogfish captures in gillnet gear, with size, sex, and environmental data taken at each station, 10.5256/f1000research.4890.d33066 (Bangley & Rulifson, 2014).", "appendix": "Author contributions\n\n\n\nCB conceived of the survey, performed field and laboratory duties, and prepared the manuscript. RR contributed to the survey design, provided expertise in spiny dogfish migration patterns, and revised the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis project was supported by internal funding from the East Carolina University Institute for Coastal Science and Policy.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors would like to acknowledge Cecilia Krahforst, Meganne Rose, Elizabeth McDonald, Debbie Lichti, Steve Licthi, Michael Flynn, Hilde Zenil, Kate Brogan, Nina Sassano, Mary Allen, Maria de Oca, Eric Schnatter, and Chris Hiltz for assistance in the field; Mike Baker, Eric Diaddorio, and Mark Keusenkothen for assistance with boating and logistical issues; and David Griffith and Hans Vogelsong for support at the institutional level.\n\n\nReferences\n\nAPNENP. Submerged aquatic vegetation – SAV. NCDENR, Albemarle-Pamlico National Estuary Partnership. (May 2014). 2008. Reference Source\n\nBangley CW, Rulifson RA: Date, time, and location of spiny dogfish captures in gillnet gear, with size, sex and environmental data taken at each station. F1000Research. 2014. Data Source\n\nBearden CM: Occurrence of spiny dogfish, Squalus acanthias, and other elasmobranchs in South Carolina coastal waters. Copiea. 1965; 1965: 378.\n\nCampana SE, Gibson AJF, Marks L, et al.: Stock structure, life history, fishery and abundance indices for spiny dogfish (Squalus acanthias) in Atlantic Canada.. Canadian Science Advisory Secretariat Research Document 2007/089. 2007. Reference Source\n\nDell’Apa A, Cudney-Burch J, Kimmel DG, et al.: Sexual segregation of spiny dogfish in fishery-dependent surveys in Cape Cod Massachusetts: potential management benefits. Trans Am Fish Soc. 2014; 143(4): 833–844. Publisher Full Text\n\nHeithaus MR, Wirsing AJ, Dill LM: The ecological importance of intact top-predator populations: a synthesis of 15 years of research in a seagrass ecosystem. Mar Freshw Res. 2012; 63(11): 1039–1050. Publisher Full Text\n\nLink JS, Garrison LP, Almeida FP: Ecological interactions between elasmobranchs and groundfish species on the northeastern U.S. continental shelf I. evaluating predation. North Am J Fish Management. 2002; 22(2): 550–562. Publisher Full Text\n\nMethratta ET, Link JS: Ontogenetic variation in habitat association for four groundfish species in the Gulf of Maine – Georges Bank region. Mar Ecol Prog Ser. 2007; 338: 169–181. Publisher Full Text\n\nNammack MF, Musick JA, Colvocoresses JA: Life history of spiny dogfish off the northeastern United States. Trans Am Fish Soc. 1985; 114(3): 367–376. Publisher Full Text\n\nRago PJ, Sosebee KA: Biological reference points for spiny dogfish. Northeast Fisheries Science Center, Reference Document 10–06, Woods Hole, MA. 2010. Reference Source\n\nRulifson RA, Moore TM: Population estimates of spiny dogfish aggregations overwintering south of Cape Hatteras, North Carolina, using an area density method. In V.F. Gallucci, G.A. McFarlane, and G.G. Bargmann, editors. Biology and management of dogfish sharks. American Fisheries Society, Bethesda, Maryland. 2009; 133–138. Reference Source\n\nRulifson RA, Cudney-Burch JE, Hemilright D: Coastal movements of spiny dogfish overwintering off the Outer Banks, NC. Completion Report, North Carolina State University, North Carolina Sea Grant, Fisheries Resource Grant Program, Grant number 08–FEG-11, Raleigh. 2012.\n\nSagarese SR, Frisk MG, Miller TJ, et al.: Influence of environmental, spatial, and ontogenetic variables on habitat selection and management of spiny dogfish in the northeast (US) shelf large marine ecosystem. Canadian J Fish Aquatic Sci. 2014; 71(4): 567–580. Publisher Full Text\n\nSchwartz FJ: Sharks, skates, and rays of the Carolinas. University of North Carolina Press, Chapel Hill, NC. 2003. Reference Source\n\nStehlik LL: Essential fish habitat source document: spiny dogfish, Squalus acanthias, life history and habitat characteristics. NOAA Technical Memorandum NMFS-NE-203. 2007. Reference Source\n\nSulikowski JA, Galuardi B, Bubley W, et al.: Use of satellite tags to reveal the movements of spiny dogfish Squalus acanthias in the western North Atlantic Ocean. Mar Ecol Prog Ser. 2010; 418: 249–254. Publisher Full Text\n\nThorpe T, Beresoff D: Determination of gillnet bycatch potential of spiny dogfish (Squalus acanthias L.) in southeastern North Carolina. Completion Report, North Carolina State University, North Carolina Sea Grant, Fisheries Resource Grant Program, Grant number 99–FEG-47, Raleigh, 2000. Reference Source\n\nThorpe T, Jensen CF, Moser ML: Relative abundance and reproductive characteristics of sharks in southeastern North Carolina coastal waters. Bull Mar Sci. 2004; 74: 3–20. Reference Source\n\nUlrich GF, Jones CM, Driggers WB, et al.: Habitat utilization, relative abundance, and seasonality of sharks in the estuarine and nearshore waters of South Carolina. In C. T. McCandless, N. E. Kohler, and H. L. Pratt, Jr., editors. Shark nursery grounds of the Gulf of Mexico and the east coast waters of the United States. American Fisheries Society, Symposium 50, Bethesda, MD. 2007; 125–139. Reference Source" }	[ { "id": "5786", "date": "10 Sep 2014", "name": "Bryan Frazier", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis research note presents observations on the temporal and spatial distribution of spiny dogfish, Squalus acanthias, in the western North Atlantic Ocean. Specifically the note documents presence and environmental data associated with the capture of spiny dogfish in the Back and Core Sound, North Carolina. While spiny dogfish have been documented later in the year (as late as June 1), associated environmental data have not been published. The findings published are of importance as they document the occurrence of mature female spiny dogfish in water temperatures above previously published thermal thresholds. Given recent management actions, these data are important as spiny dogfish may occur in their southern range well outside of periods where they are sampled.These data are clear and concise and appropriate for publication as a research note. The data are straightforward, and I recommend indexing the article, although the authors should consider the following minor revisions. Abstract: Report ranges and mean lengths for spiny dogfish captured. Length at maturity should state length at 50% maturity. Significant digits for depth should be reported to the nearest tenth of a meter, this should also be corrected throughout the manuscript.Methods: Paragraph two, the authors should clarify if total length measurements are natural total length, or stretch total length. If they are estimating maturity based on length this could affect estimates.Results: Significant digits need to be corrected, again depths should reported to the nearest tenth of a meter, also DO significant digits are reported to the nearest tenth in one instance and the nearest one hundredth in the next.For capture events it isn't necessary to report additional catch (batoids) as these data do not contribute to the note.Discussion: If temperature data are available for the Rulifson et al. (2012) reported June encounters of spiny dogfish, these data should be reported.For Nammack et al reference, report what metric of length at maturity this is (median or length at 50% maturity)", "responses": [] }, { "id": "6101", "date": "22 Sep 2014", "name": "Walter Bubley", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an informative manuscript detailing previously unrecorded maximum water temperatures in which Spiny Dogfish, Squalus acanthias, have been encountered during a standardized survey off the coast of North Carolina. I believe though that the authors need to pare back the content in the methods section to that relevant to the main focus of the paper and not overstate the findings as they relate to habitat use within the estuary in the results and discussion sections. The information obtained in this manuscript has importance in documenting previously unknown temperatures but the authors need to understand that this is the importance and not try to interpret minimal data for habitat use.AbstractRemove sentence beginning with \"Stations where dogfish...\" as there are not enough samples to make any sort of inference to estuary or habitat useMethodsRemove the sentence in the first paragraph beginning with \"Sampling locations were chosen...\" as this is not relevant to the capture of the Spiny Dogfish.Remove the mention and description of longline gear use in the second paragraph, as the spiny dogfish were all caught using gillnets. While the survey may have utilized both gear types, the Spiny Dogfish focused on in this paper were not caught with longline.Remove all sentences beginning with \"Distance from the nearest inlet...\" through the remainder of the second paragraph, as this information is not utilized in the analysis.Remove the sentences beginning with \"All batoids were identified...\" through the end of the paragraph as this is not relevant to the topic of this manuscript.ResultsRemove all mention of other species captured concurrently with Spiny Dogfish.Remove sentences referring to capture site in relation to inlets and seagrass areas. There is not enough information to do analysis in regards to habitat use.DiscussionRemove the sentence in the fourth paragraph beginning with \"Spiny dogfish feed primarily on...\" as this is way too speculative based on the catch of a few harvest fish in one net that also caught spiny dogfish.Remove the fourth paragraph as there are too few samples collected to make any inferences about habitat use for this manuscript.In general, the manuscript provides useful information in regards to new maximum temperatures recorded for spiny dogfish catch, but I want to emphasize that I would caution the authors from making too many speculative assertions based on limited catch at these temperatures.", "responses": [] } ]	1	https://f1000research.com/articles/3-189
https://f1000research.com/articles/3-239/v1	09 Oct 14	{ "type": "Research Article", "title": "Predictive factors for difficult mask ventilation in the obese surgical population", "authors": [ "Davide Cattano", "Anastasia Katsiampoura", "Ruggero M. Corso", "Peter V. Killoran", "Chunyan Cai", "Carin A. Hagberg", "Anastasia Katsiampoura", "Ruggero M. Corso", "Peter V. Killoran", "Chunyan Cai", "Carin A. Hagberg" ], "abstract": "BackgroundDifficult Mask Ventilation (DMV), is a situation in which it is impossible for an unassisted anesthesiologist to maintain oxygen saturation >90% using 100% oxygen and positive pressure ventilation to prevent or reverse signs of inadequate ventilation during mask ventilation. The incidence varies from 0.08 – 15%. Patient-related anatomical features are by far the most significant cause. We analyzed data from an obese surgical population (BMI> 30 kg/m2) to identify specific risk and predictive factors for DMV.MethodsFive hundred and fifty seven obese patients were identified from a database of 1399 cases associated with preoperative airway examinations where mask ventilation was attempted. Assessment of mask ventilation in this group was stratified by a severity score (0-3), and a step-wise selection method was used to identify independent predictors. The area under the curve of the receiver-operating-characteristic was then used to evaluate the model’s predictive value. Adjusted odds ratios and their 95% confidence intervals were also calculated.ResultsDMV was observed in 80/557 (14%) patients. Three independent predictive factors for DMV in obese patients were identified: age 49 years, short neck, and neck circumference 43 cm. In the current study th sensitivity for one factor is 0.90 with a specificity 0.35. However, the specificity increased to 0.80 with inclusion of more than one factor.ConclusionAccording to the current investigation, the three predictive factors are strongly associated with DMV in obese patients. Each independent risk factor alone provides a good screening for DMV and two factors substantially improve specificity. Based on our analysis, we speculate that the absence of at least 2 of the factors we identified might have a significant negative predictive value and can reasonably exclude DMV, with a negative likelihood ratio 0.81.", "keywords": [ "Airway Management", "Mask Ventilation", "Obesity", "Obstructive Sleep Apnea" ], "content": "Introduction\n\nBag mask ventilation commonly precedes the establishment of a secure airway by endotracheal intubation. However, the degree of difficulty encountered is variable1–4, with the incidence of Difficult Mask Ventilation (DMV) varying from 0.08–15% depending on the criteria used for the definition. The American Society of Anesthesiologists’ (ASA) original definition recognized DMV as a situation where it is not possible for the unassisted anesthesiologist to maintain the oxygen saturation > 90% using 100% oxygen and positive pressure ventilation, or to prevent or reverse signs of inadequate ventilation5. Subsequently, many other definitions have evolved taking into account patient-independent factors that contribute to DMV, such as provider--and equipment-related factors5. Moreover, as an effort to overcome subjective definitions, several grading scales have been proposed, including Adnet’s and Han’s scales1,6.\n\nIn the face of DMV, critical hypoxemia may rapidly ensue and emphasizes the need for proper identification of risk factors during the preoperative assessment. Obese patients remain one of the most challenging patient populations for airway management7–9, with difficulties arising due to both anatomical features and functional changes10,11. Current protocols for preoperative evaluation focus not only on anatomic characteristics, but also on the identification of systemic features that are associated with airway obstruction and physiologic disarrangements, such as obstructive sleep apnea syndrome (OSA)8,12. For instance, in the general surgical population, a history of OSA has been found to be an independent risk factor of impossible mask ventilation13,14, and patients with a high BMI have a high risk for OSA12. However, despite the known association between DMV, obesity, and OSA, there are no established predictive criteria, nor a simple scoring system which could predict DMV in the obese population.\n\nIn the present investigation, we primarily aimed to identify specific risk and predictive factors for difficult mask ventilation in obese patients and secondarily we attempted to correlate history and predicted factors related to OSA with DMV. We performed a retrospective analysis based on an existing database14.\n\n\nMethods\n\nA retrospective investigation was performed to identify predictive markers of DMV in obese patients at Memorial Hermann Hospital-Texas Medical Center utilizing an existing database of airway assessment and airway management records4,14 : 1399 anesthetics were identified where both mask ventilation was attempted and a pre procedure airway evaluation was documented. Of these, 557 obese patients were identified and included for analysis. The preoperative assessment utilized a dedicated airway assessment form14 which included Mallampati pharyngeal classification (modified by Samsoon and Young)15, inter-incisor gap and thyromental distance (cm) measured with the neck extended, sternomental distance, BMI, neck circumference (cm) measured at the level of the thyroid cartilage, dentition status, presence of facial hair, facial or neck trauma, nasal deficiencies, neck mobility grade (which was divided into three categories according to the mouth-occiput distance), diagnosis of OSA according to patient history, perceived short neck, history of difficult intubation, and cervical spine abduction. Due to the retrospective nature of the study we were able to assess the OSA status only by the patient history. The degree of DMV classified by the provider performing the case by a severity score1: 0 = easy, 1 = oral airway used, 2 = two handed ventilation and 3 = extraglottic device required. Based on the severity, mask ventilation was considered True DMV if the ease of mask ventilation was graded as 2 or 3 and False DMV if it was graded as 0 or 1. During attempts at mask ventilation, all obese patients were placed in the head elevated laryngoscopy position and the operating room table was titled in the reverse Trendelenburg position. Vital signs were monitored according to ASA standard general anesthesia monitoring. Neuromuscular blocking agent utilization and/or the time of administration, dosage and reason for administration was not captured in the source database and therefore not included in this retrospective investigation.\n\n\nStatistical analysis\n\nStatistical analyses was performed using SAS 9.3 (SAS Institute, Cary, NC, USA). A p-value <0.05 was considered significant. Obese patients with or without DMV were compared. Values were reported as mean ± standard deviation for continuous variables and frequency (percentage) for categorical variables for all preoperative patient characteristics. First, a univariate comparison between patients with or without DMV was performed using two sample t-test for continuous variables and Chi-square test or Fisher exact test, as appropriate, for categorical variables. Age was dichotemized based on a threshold of 49 years and neck circumference of 43 (cm), based on recognized risk threshold7. All variables with a p-value <0.20 in univariate analysis were entered into a multivariate logistic regression model. Stepwise selection method was used to identify independent predictors of DMV. All variables that were statistically significant with a p < 0.05 were established as independent predictors. Age and neck circumference were dichotomized according to clinical suggestions, using the optimal cut-off value identified by maximizing the sum of sensitivity and specificity for the primary outcome to obtain the best accuracy. In addition, the area under the curve of the receiver-operating-characteristic was calculated to evaluate the resulting model’s predictive value. The adjusted odds ratios and their 95% confidence intervals were also calculated.\n\n\nResults\n\nA total of 557 cases of attempted mask ventilation were recorded in obese patients, as shown in Table 1, of which 78 were considered to be DMV (14.3%). Patient characteristics and statistical correlations between DMV and preoperative variables are presented in Table 2.\n\nDefine DMV=True if MVEase=2,3 and DMV=False if MVEase=0,1.\n\nNR: not reported due to zero cells. Values are reported as mean±SD and frequency (percentage).\n\nBased on a univariate analysis, a total of 6 factors were identified with a p value < 0.05 including: age, gender, neck circumference, absence of teeth, short neck (subjective) and ΟSA (suspected or diagnosed). Thresholds used were based on clinical suggestions. Age was dichotomized based on a threshold of 49 years of, and neck circumference based on, 43 cm. Incorporation of these 6 factors into a multivariate logistic regression model identified 3 independent predictive factors for DMV in obese patients. The model used step-wise selection and identified age ≥ 49 years, short neck, and neck circumference ≥ 43 cm (Table 3) as statistically significant. OSA, gender, and absence of teeth were not considered significant in the multivariate model.\n\nAlthough a total of 3 risk factors were identified, no individual subject had more than 2 risk factors. The 3 independent risk factors identified were then applied to all cases where DMV was encountered to evaluate a predictive model for DMV in obese patients. The sensitivity, specificity, likelihood ratios, and predictive values were progressively calculated for patients with different numbers of risk factors. The adjusted odds ratios were analyzed (Table 4).\n\nLikelihood ratio positive=Sensitivity/(1-Specificity) Likelihood ratio negative=(1-Sensitivity)/Specificity.\n\nTable 4 displays the sensitivity and specificity if we use the given value of the number of risk factors possessed by patients as a cut-off to classify DMV. For example, when we use number of risk factors at 1 as a cut-off, i.e., any patients with >=1 risk factors will be classified as DMV=1 and any patients with <1 risk factors will be classified as DMV=0, the sensitivity will be 0.90 and specificity will be 0.34. Cut-off at 1,2 are calculated and displayed.\n\nA ROC curve (Figure 1) evaluating the sensitivity and specificity of preoperative independent risk factors for DMV for BMI>30 kg/m2 patients was calculated. The model’s c-statistic score was 0.65 with 95% CI of 0.59 to 0.70. The sensitivity for one factor is 0.90 with a specificity of 0.35. However with more than one factor, the specificity increased to the level of 0.80.\n\nThree independent predictors for difficult mask ventilation were identified using logistic regression: age of 49 yr or older, NeckCirc of 43 or greater, and Short Neck. The area under the curve was 0.65 (95% confidence interval: 0.59 – 0.70).\n\n\nDiscussion\n\nWe performed a retrospective analysis based on a database of airway assessment and airway management records collected at Memorial Hermann Hospital-Texas Medical Center, a tertiary care center. Our study focused in stratification and the identification of DMV predictive factors in a surgical population of obese patients, while recently we reported DMV in the general population4.\n\nIn our cohort, the incidence of DMV in obese patients was 14%. These findings are consistent with previous reports by Leoni and Kheterpal7,13. In their study, Leoni et al. reported that the incidence of DMV is significantly higher in obese patients compared to the general surgical population7. We also compared the incidence of DMV in the obese population to the general surgical population, confirming a frequency of 14% and 8.84,9 respectively. The finding emphasizes the different risk stratification of DMV in the obese patients. Interestingly, in our obese surgical population OSA was as frequent as 24%, while in other studies the prevalence of OSA among bariatric surgery patients reaches up to 70% and, in the general population, is approximately 20%16–18.\n\nIn the present investigation, the statistical analysis identified 3 novel independent predictive markers for DMV in obese population: (a) age ≥ 49 years, (b) neck circumference ≥ 43 cm, and (c) perceived short neck. In the general population 7 risk factors were previously identified, of which OSA and BMI were two of them4: interestingly the latter together with facial hair and history of difficult intubation were not present in the current model. This could be attributed to the reduction of the sample size, to the specific characteristic of obese patient (which are not necessarily at increased risk of difficult intubation)19 or the effect of the stratification used which could mask the effect of BMI and OSA.\n\nBased on our analysis, we speculate that the absence of at least 2 of the factors we identified might have a significant negative predictive value and can reasonably exclude DMV, with a negative likelihood ratio 0.81. To our knowledge, this is the first time that short neck and age ≥ 49 years are recognized as risk factors for DMV specifically in obese patients; however this is not totally unexpected. According to Langeron et al., age >55 years is correlated with DMV in the general population20, thus it seems reasonable that age would be a risk factor for DMV in the obese population as well. Shah et al. consider short and thick neck as an independent risk factor in the general population21. Neck circumference could be correlated to anatomical and physiological changes due to obesity that may increase the airway obstruction. Indeed the increased neck circumference is reflecting the presence of excessive palatal and pharyngeal soft tissue which intensifies the collapse of oropharynx during muscle relaxation. As a result increased neck circumference can make mask ventilation more difficult22,23.\n\nNumerous prospective and retrospective clinical studies examined the correlation of patient-dependent and patient-independent characteristics, along with DMV, in the general surgical population8,20,24, and led to the identification of several predictive factors for DMV. Specifically, Langeron et al. (as previously stated), Yildiz et al. and Kheterpal et al. demonstrated that increased BMI, history of snoring or OSA, as well as age ≥ 55 years are risk factors for DMV in the general surgical population13,20,24. Additional factors in these studies included the presence of beard, Mallampati classification of III or IV, limited mandibular protrusion test, male gender, and airway masses or tumors. In our investigation, a total of 6 predictive markers of DMV were identified. However, Mallampati classification, limited mandibular protrusion and male sex did not reach significant correlation to DMV (step-wise analysis). All these findings are summarized in Table 5.\n\nAdjusted odds ratios with 95% Confidence intervals and P values are noted respectively.\n\nLast and with our surprise, OSA was not an independent risk factor for DMV in our cohort: this could be explained by the overlap of OSA predictive value with other factors, such as neck circumference, which has been shown to correlate with OSA25.\n\nFew comments need to be reserved for the limitations of the present investigation. First, resident physicians were mostly involved in the study and we assumed that all anesthesiology residents had similar educational skills, based on our recent study9. Another limitation is the fact that the report regarding DMV is based on the subjective nature of the DMV definitions. Third, stepwise selection was sample dependent and may artificially enhance the performance of the model. Fourth, the retrospective nature of our data selection could contribute to bias in this study. Lastly, mask ventilation was assumed to be assessed as per current practice after induction and before muscle relaxation, yet the absence of an objective measure in the study about the status of paralysis and the use of muscle relaxant before or after the assessment of the mask ventilation could have partially affected the results.\n\n\nConclusion\n\nIn conclusion, in the present study we demonstrated that (a) age ≥ 49 years, (b) neck circumference ≥ 43 cm, and (c) short neck (perceived) are strongly associated with DMV in obese patients. Thus, we suggest that these patient-dependent factors should be included in the pre-operative assessment to better predict DMV in the obese population. Each one used singularly may provide an efficacious screening tool, while the association of 2 of them may be used to improve specificity. Since the prevalence of obese patients in the surgical population is increasing exponentially, further investigation is warranted that may elucidate the association of (1) patient-derived anatomical and functional characteristics, (2) physician-derived characteristics and (3) equipment characteristics with DMV in obese patient.\n\n\nData availability\n\nData have been obtained from databases at the Memorial Hermann Hospital, Texas Medical Center, Houston, IRB approval HSC-MS-07-0144. The author can support applications to the Institutional Board to make the data accessible upon individual request. Please forward your requests to Davide Cattano.", "appendix": "Author contributions\n\n\n\nDavide Cattano: study design, study monitoring, data analysis, data interpretation, manuscript preparation\n\nAnastasia D. Katsiampoura: manuscript preparation, data analysis, data interpretation\n\nRuggero M. Corso: data interpretation, manuscript preparation\n\nPeter V. Killoran: study monitoring, data collection, data analysis, data interpretation\n\nChunyan Cai: data analysis, manuscript preparation\n\nCarin A. Hagberg: study design, study monitoring, manuscript preparation\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was sponsored by the Foundation in Anesthesia, Education and Research as the 2007 FAER Education Grant. Dr. Carin A. Hagberg was the Principle Investigator and Dr. Davide Cattano, the Co-Investigator.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nHan R, Tremper KK, Kheterpal S, et al.: Grading scale for mask ventilation. Anesthesiology. 2004; 101(1): 267. PubMed Abstract \| Publisher Full Text\n\nKheterpal S, Han R, Tremper KK, et al.: Incidence and predictors of difficult and impossible mask ventilation. Anesthesiology. 2006; 105(5): 885–91. PubMed Abstract \| Publisher Full Text\n\nBenumof JL: Management of the difficult adult airway. With special emphasis on awake tracheal intubation. Anesthesiology. 1991; 75(6): 1087–110. PubMed Abstract \| Publisher Full Text\n\nCattano D, Killoran PV, Cai C, et al.: Difficult mask ventilation in general surgical population: observation of risk factors and predictors. [v1; ref status: approved1, http://f1000r.es/47z]. F1000Research. 2014; 3: 204. Publisher Full Text\n\nPractice guidelines for management of the difficult airway. A report by the American Society of Anesthesiologists Task Force on Management of the Difficult Airway. Anesthesiology. 1993; 78(3): 597–602. PubMed Abstract \| Publisher Full Text\n\nAdnet F: Difficult mask ventilation: an underestimated aspect of the problem of the difficult airway? Anesthesiology. 2000; 92(5): 1217–8. PubMed Abstract \| Publisher Full Text\n\nLeoni A, Arlati S, Ghisi D, et al.: Difficult mask ventilation in obese patients: analysis of predictive factors. Minerva Anestesiol. 2014; 80(2): 149–57. PubMed Abstract\n\nKheterpal S, Martin L, Shanks AM, et al.: Prediction and outcomes of impossible mask ventilation: a review of 50,000 anesthetics. Anesthesiology. 2009; 110(4): 891–7. PubMed Abstract \| Publisher Full Text\n\nCattano D, Killoran PV, Iannucci D, et al.: Anticipation of the difficult airway: preoperative airway assessment, an educational and quality improvement tool. Br J Anaesth. 2013; 111(2): 276–85. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJense HG, Dubin SA, Silverstein PI, et al.: Effect of obesity on safe duration of apnea in anesthetized humans. Anesth Analg. 1991; 72(1): 89–93. PubMed Abstract \| Publisher Full Text\n\nBenumof JL: Obesity, sleep apnea, the airway and anesthesia. Curr Opin Anaesthesiol. 2004; 17(1): 21–30. PubMed Abstract \| Publisher Full Text\n\nCorso RM, Petrini F, Buccioli M, et al.: Clinical utility of preoperative screening with STOP-Bang questionnaire in elective surgery. Minerva Anestesiol. 2013; 80(8): 877–84. PubMed Abstract\n\nKheterpal S, Healy D, Aziz MF, et al.: Incidence, predictors, and outcome of difficult mask ventilation combined with difficult laryngoscopy: a report from the multicenter perioperative outcomes group. Anesthesiology. 2013; 119(6): 1360–9. PubMed Abstract \| Publisher Full Text\n\nKilloran PV, Maddukuri V, Altamirano A, et al.: Use of a comprehensive airway assessment form to predict difficult mask ventilation. Anesthesiology. 2011; A442. Reference Source\n\nSamsoon GL, Young JR: Difficult tracheal intubation: a retrospective study. Anaesthesia. 1987; 42(5): 487–90. PubMed Abstract \| Publisher Full Text\n\nLopez PP, Stefan B, Schulman CI, et al.: Prevalence of sleep apnea in morbidly obese patients who presented for weight loss surgery evaluation: more evidence for routine screening for obstructive sleep apnea before weight loss surgery. Am Surg. 2008; 74(9): 834–8. PubMed Abstract\n\nLee W, Nagubadi S, Kryger MH, et al.: Epidemiology of Obstructive Sleep Apnea: a Population-based Perspective. Expert Rev Respir Med. 2008; 2(3): 349–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGami AS, Caples SM, Somers VK: Obesity and obstructive sleep apnea. Endocrinol Metab Clin North Am. 2003; 32(4): 869–94. PubMed Abstract \| Publisher Full Text\n\nBrodsky JB, Lemmens HJ, Brock-Utne JG, et al.: Morbid obesity and tracheal intubation. Anesth Analg. 2002; 94(3): 732–6. PubMed Abstract \| Publisher Full Text\n\nLangeron O, Masso E, Huraux C, et al.: Prediction of difficult mask ventilation. Anesthesiology. 2000; 92(5): 1229–36. PubMed Abstract \| Publisher Full Text\n\nShah PN, Sundaram V: Incidence and predictors of difficult mask ventilation and intubation. J Anaesthesiol Clin Pharmacol. 2012; 28(4): 451–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWatanabe T, Isono S, Tanaka A, et al.: Contribution of body habitus and craniofacial characteristics to segmental closing pressures of the passive pharynx in patients with sleep-disordered breathing. Am J Respir Crit Care Med. 2002; 165(2): 260–5. PubMed Abstract \| Publisher Full Text\n\nIsono S: Obstructive sleep apnea of obese adults: pathophysiology and perioperative airway management. Anesthesiology. 2009; 110(4): 908–21. PubMed Abstract \| Publisher Full Text\n\nYildiz TS, Solak M, Toker K: The incidence and risk factors of difficult mask ventilation. J Anesth. 2005; 19(1): 7–11. PubMed Abstract \| Publisher Full Text\n\nHiremath AS, Hillman DR, James AL, et al.: Relationship between difficult tracheal intubation and obstructive sleep apnoea. Br J Anaesth. 1998; 80(5): 606–11. PubMed Abstract \| Publisher Full Text" }	[ { "id": "6608", "date": "31 Oct 2014", "name": "Basem Abdelmalak", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this very interesting manuscript that focuses on studying difficult mask ventilation in obese patients. This manuscript highlights an important safety consideration when caring for this patient population. The authors have appropriately discussed the limitations of this study. Of those, the most important is the retrospective nature of the study. Another limitation to consider is the lack of data on the use of muscle relaxants: how much and its timing relative to the assessment of the ventilation difficulty, etc. As one might imagine, there may have been variability related to the ongoing controversy of the administration of muscle relaxants either immediately after induction, or a bit delayed till confirmation of the ability to mask ventilate.As we acknowledge such limitations and thus their impact on the validity of these results, we should keep in mind that the resulting increased awareness of these predictors will likely increase the likelihood of a thorough airway exam and making the right decision in managing such airways. As per the most recent ASA difficult airway algorithm (2013) a thorough airway evaluation will aid in deciding the safest pathway taken inclusive of the following factors: invasive vs, non-invasive, awake vs. asleep, videolaryngoscopy as first approach vs. DL, and finally maintaining spontaneous ventilation vs. muscle relaxation.", "responses": [ { "c_id": "1055", "date": "31 Oct 2014", "name": "Davide Cattano", "role": "Author Response", "response": "Dr Abdelmalak comments are very appreciated, particularly considering his airway management expertise. The points underlined are significant to strenght discussion about our findings. We would like to comment about the muscle relaxation utilization: as clinical standard at our institution, muscle relaxant administration, the majority of the times, follows bag mask ventilatilability confirmation, but it is also true that several anesthesiologists actually utilize a different pattern (which it has been predicated by expert society as well).Another important point pertains the predictors. It is interesting to map the risk predictors as per different studies have identified, and knowing the clinical practice pressure we are exposed to, making an effort for the few that seems to have a significant impact may be worth more than being distracted by others.The last point is related to airway devices and techniques. As standard or routine utilization of certain devices becomes more common, the difficulty of airway management is also evolving. We cannot disregard in this sense the work of Caldiroli and Cortellazzi for instance on the utilization of Glidescope as primary laryngoscopy device: such usage has prompted a revisitation of the El-Ganzouri score, highlighting newer usage of predictive factors because of modification of airway difficulty and outcomes.Lastly, It is reassuring that several investigations, which ours is one, particularly in different patient populations, are confirming in parallel same findings." } ] }, { "id": "6376", "date": "05 Nov 2014", "name": "Peter Szmuk", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this manuscript and my apologies for the long response time.This manuscript comes from one of the world leading research centers on difficult airway and deals with a very important and possible life saving topic: the difficult mask ventilation (DMV). Based on a retrospective analysis the authors found that three factors (age > 49, neck circumference > 43 and short neck) are strong predictors for DMV in obese patients and thus, should be included in a pre-operative assessment. Despite the retrospective nature of the study, this screening tool should make an important contribution to the predictability of DMV especially in the obese patients. I would encourage the authors to expand their work into a prospective study.Minor comments: Methods section: 1399 anesthetics were identified to have both mask ventilation and airway evaluation. Please specify over what period and/or how many patients.Please define “obese” patients and clarify the term “nasal deficiencies”. I am surprised that the dentition status was not one of the factors involved in the DMV. Is that due to the fact that very few patients in your group are edentulous? This might be different in other geographical areas. Could you comment on that? Ethnicity and body fat distribution would be another factor to consider in a future study. Obese patients with predominant abdominal fat distribution might have normal neck and airway as compared to those with an equally distributed fat habitus. Finally, one of the study limitations noted in the discussion section mentioned the skills level of the residents participating in these cases. This is a valid concern but I would also be interested to see if other factors related to the provider might play a role in DMV. From my clinical observation I noticed that providers with small hands have more difficulty with mask ventilation of a large, obese patient. I wander if anybody looked into this association.", "responses": [ { "c_id": "1063", "date": "05 Nov 2014", "name": "Davide Cattano", "role": "Author Response", "response": "We acknowledge Dr Szmuk comments, and the discussion resulting could further strengthen the data reported. The study was undergone over a period of 18 months, and a selected review of all records with pre- and post intervention data available constituted the cohort analysis. The definition of obesity is the WHO, BMI greater and equal of 30 (kg/m2). We recognize the limitations of a retrospective review and the deficiency in some of the definition, which are clinical indicators though in real world airway management. Indeed in a previous investigation we commented on the inevitable fact that certain predictors remains subjective and dependent on operator assessment. It is interesting that many classification based on standardized definition performs at most with 65% reliability. We agree with Dr Szmuk that body fat distribution is indeed perceived as an important factor. It is interesting that in other studies BMI as low as 26 were found to be associated (general population) and that in European and Asia based study the incidence of DMV would be variable based on other anatomical markers and different body weight, maybe confirming Dr Szmuk's observation." } ] }, { "id": "6375", "date": "07 Nov 2014", "name": "Mirsad Dupanović", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI would like to compliment Dr. Cattano and his colleagues for undertaking the investigation on this important topic. Their results confirmed most of the risk factors that other investigators have also found may make mask ventilation difficult (studies cited in the reference section of this manuscript). However, Cattano et al. have attempted to go a step further and identify as to how many risk factors need to be present for mask ventilation to be difficult. They had a partial success in that venture. The definition of difficult mask ventilation in this study seems very reasonable and I hope it gets wide acceptance among those that manage airway.I agree with Dr. Doyle's comment that it needs to be re-emphasized that this was a retrospective study and these results should be re-tested in a well designed prospective study.", "responses": [] } ]	1	https://f1000research.com/articles/3-239
https://f1000research.com/articles/3-236/v1	07 Oct 14	{ "type": "Software Tool Article", "title": "Niche Genetic Algorithms are better than traditional Genetic Algorithms for de novo Protein Folding", "authors": [ "Michael Scott Brown", "Tommy Bennett", "James A. Coker", "Michael Scott Brown", "Tommy Bennett" ], "abstract": "Here we demonstrate that Niche Genetic Algorithms (NGA) are better at computing de novo protein folding than traditional Genetic Algorithms (GA). Previous research has shown that proteins can fold into their active forms in a limited number of ways; however, predicting how a set of amino acids will fold starting from the primary structure is still a mystery. GAs have a unique ability to solve these types of scientific problems because of their computational efficiency. Unfortunately, GAs are generally quite poor at solving problems with multiple optima. However, there is a special group of GAs called Niche Genetic Algorithms (NGA) that are quite good at solving problems with multiple optima. In this study, we use a specific NGA: the Dynamic-radius Species-conserving Genetic Algorithm (DSGA), and show that DSGA is very adept at predicting the folded state of proteins, and that DSGA is better than a traditional GA in deriving the correct folding pattern of a protein.", "keywords": [ "Proteins are one of the basic building blocks of all life and we have learned much about them since they were ‘discovered’ about 200 years ago1", "including their shapes", "functionality", "and uses. However", "there are still many basic questions such as how proteins are able to transform from a useless linear assemblage of amino acids (primary structure) into a functional three-dimensional native structure", "that remain unanswered2. The ability to accurately predict the functional form of a protein from its primary sequence would revolutionize many fields and has long been considered a ‘holy grail’ in Life Sciences research3." ], "content": "Introduction\n\nProteins are one of the basic building blocks of all life and we have learned much about them since they were ‘discovered’ about 200 years ago1, including their shapes, functionality, and uses. However, there are still many basic questions such as how proteins are able to transform from a useless linear assemblage of amino acids (primary structure) into a functional three-dimensional native structure, that remain unanswered2. The ability to accurately predict the functional form of a protein from its primary sequence would revolutionize many fields and has long been considered a ‘holy grail’ in Life Sciences research3.\n\nThe solution to the problem of computationally determining how proteins fold will involve multiple disciplines of science making it a very interesting topic to address. At its heart, it is a biochemical issue, rooted in both geometry and physics, which is faced by every cell on Earth. In Mathematical terms, it is an application of the ‘self-avoiding walk’ problem4 with some additional constraints. Since we know that there are many physical constraints on achieving a properly folded protein, it is also an NP-hard problem5 and therefore highly applicable to Computer Science.\n\nProteins are made up of a sequence of amino acids, of which there are many types but only 20 are typically used in biological proteins6. Each amino acid type can be placed into one of two categories: hydrophilic (P) and hydrophobic (H). While a protein’s primary sequence dictates the ordering of the amino acids, it must fold into a three-dimensional structure to be active7. Therefore, the goal of solving how proteins fold computationally is to determine the folding pattern of any protein starting from the primary sequence.\n\nFor the last 100 years, the two most employed methods for determining the folding pattern of a given protein are x-ray crystallography8 and nuclear magnetic resonance (NMR)9. Both can provide high resolution images of the folded-state of a protein but rely on the ability of scientists to purify the protein of interest to concentrations of 1 molar (NMR) to greater than 10 molar (x-ray crystallography), which is not an easy task. The x-ray crystallography method is further complicated by the need to determine the conditions for the growth of crystals of the protein and then waiting for those crystals to grow to a usable size and dimension10. The NMR method requires less purified protein, compared to x-ray crystallography; however, it is limited by the size of protein that can be analyzed10. Both of these more traditional methods also require a significant investment of time in order to achieve the folding pattern of a protein. Despite the significant time investment, these methods have persisted due to the fact that they are a very reliable way to determine protein structure. However, with the recent explosion of genomes being sequenced, which has resulted in the discovery of a plethora of new proteins and protein families, newer methods that will reliably determine the folding pattern of a protein in a shorter amount of time are called for11. One of these methods, de novo protein folding, uses only the primary sequence of a protein and a set of computer algorithms to determine its active, folded form. This may seem overly daunting at first, since even a relatively small protein can have a nearly infinite number of possible folding patterns. However, over the past 50–60 years biochemists have determined that the way a protein folds is quite conserved in a protein family12 and that chemical/physical forces significantly reduce the number of ways a protein can fold13–15. These two findings are very important and have the very real implication that computers can be used to predict the active, folded forms of a protein in a very short period of time.\n\nOne method often used to computationally determine how proteins fold is a Genetic Algorithm (GA). GAs are a type of optimization algorithm that models biological selection16,17 and are part of a family of optimizations algorithms called Heuristics18, which attempts to solve a problem by determining a solution and iteratively making the solution better. GAs have been very good at optimization of large complex domains, are a product of the field of Artificial Intelligence and can theoretically solve any problem that can be represented as the optimization of a continuous function. The theory behind GAs states that after many generations the intermediate solutions will eventually converge upon the correct answer19. Even in cases where GAs could not fully solve the problem, the answer produced was valuable, which is an aspect of GAs that makes them superior to other algorithms.\n\nAt their core, GAs model biological selection and provide multiple possible answers termed individuals, which are comprised of a string of characters. The GA begins by randomly generating a number of individuals (first generation) and then goes through multiple iterations of selection, crossover and mutation. In selection, pairs of individuals are picked for crossover. Individuals with higher fitness, as determined by a fitness function, are given an increased probability of being selected and an individual can be selected for crossover multiple times. In crossover, two individuals are picked and each is broken into two substrings at a randomly selected position that is at the same position in the string of characters for both thereby creating two new individuals. In mutation, the value of some of the characters in each individual can change based on a probability parameter. The processes described above results in a new generation of individuals and the process then repeats using the new generation (Table 1).\n\nAlthough GAs are good at solving the optimization of a continuous function, they have a difficult time solving multi-optima problems20. When multiple optima exist a traditional GA will often locate only one optimum and there is no guarantee that it is the global optimum and not a local one. To overcome this problem, specialized GAs, called Niche Genetic Algorithms (NGA), have been developed that can locate multiple optima20,21. There are a number of NGAs including De Jong22, Crowding Clustering Genetic Algorithm (CCGA)23 and Species Conserving Genetic Algorithm (SCGA)24. One NGA has been shown to be especially adept at solving problems with multiple optima21. It is called the Dynamic-radius Species-conserving Genetic Algorithm (DSGA)20 and is basically a modification of the SCGA24. DSGA enhances the traditional GA by the addition of seeds, a Tabu List20 and the ability to change the radius. A seed is a locally strong individual based upon some radius that is identified in each iteration of the loop and conserved (i.e. propagated into the next generation by replacing a locally weak individual). A Tabu List’s function, whose name comes from the Tabu Search25, is to store strong candidates for the global optima, which is determined by the Reevaluation Loop Count (RLC) and the Convergence Limit (CL).\n\nHere we show that protein folding is a multi-optima problem and as a result NGAs are better suited for a solution. The DSGA has not previously been applied to the immense task of de novo protein folding. Therefore, as a proof of concept, we have shown two important results below: (1) the DSGA is very adept at predicting the folded state of proteins, which was shown by selecting a 20 amino acid protein and modeling the folds and all possible combinations; (2) the DSGA is better than a traditional GA and better able to derive the correct folding pattern of a protein. Below we present some preliminary testing data and have provided the source code, which is available for download at Zenodo.org (https://zenodo.org/record/11902).\n\n\nMaterials and methods\n\nEach individual is evaluated from the most to the least fit. If no other seeds exist within the radius (r) of the individual then the individual is a seed. In the Seed Conservation method each seed will replace an individual in the newly created generation. If there are individuals in the next generation within r of the seed, the seed will replace the weakest of these individuals. If there are no individuals within r of the seed in the next generation, the seed will replace the globally weakest individual. But these seeds have to re-compete to be seeds in the next generation.\n\nThe Tabu List stores potential candidates for the global optima. As individuals are put on the Tabu List, the DSGA attempts to seek optima in other locations by using a Shared Fitness. Shared Fitness will decrease the fitness of an individual if it is too close to individuals on the Tabu List. This encourages exploration in other areas of the domain. The Shared Fitness function is defined in equation 1.\n\n\n\nIn equation 1, mi is defined by equation 2 where TLj is the jth individual on the Tabu List, Length(i) is the number of characters in individual i and Distance(i, TLj) is the distance between individual i and individual TLj.\n\n\n\nThe final term in the Shared Fitness equation is + 1. Individuals with a fitness of zero have no chance of being selected for crossover. By incrementing all Shared Fitness values by one, this gives these individuals a chance at selection and propagation into later generations.\n\nFor this study, we selected chromosomal difference using Equation 3 below to calculate the distance between two individuals (i1 and i2).\n\n\n\nThis function determines how fit an individual is in relation to the fold it has adopted. The value of this function is determined by calculating the Free Energy and the algorithm prioritizes individuals with a greater ability to fold spontaneously (i.e. low value for Free Energy). Here, we calculated Free Energy by summing all of the possible contacts between adjacent, but not neighboring, hydrophobic amino acids as has been done previously26. The free energy between any two amino acids (i and j) can be found using the following formula:\n\n\n\nThe free energy (E) for a protein can be found by summing the free energy between all of the amino acids as follows:\n\nE = Σ Δrijεij\n\n\n\nAlthough proteins are three dimensional structures, it is common to use two dimensions. Using two dimensions reduces the search space in the domain. Issues with the algorithms can be addressed and future research can be published using three-dimensional models. This research uses a two dimensional model for protein folding.\n\nIn order to model protein folding, a simple method was selected where each gene of an individual has a value of zero, one, two or three. In our method, a zero, one, two or three denotes placing the next amino acid above, right, below or left the previous one, respectively. This method allows for greater simplicity and saves computing time as the number of genes needed in the individual is one less than the number of amino acids in the protein.\n\nIn some cases the model may produce a folding that isn’t physically possible (i.e. two amino acids occupying the same space). For example, the series of genes ‘13’ would not be physically possible as the second amino acid would be over top of the first. To handle these cases we employ a method we titled the Keep Going method. When directed to place an amino acid in a location that is already occupied, the algorithm will look for other positions to place it using a predictable pattern. If the folding sequence indicates placement of an amino acid in an occupied position, the algorithm will place it in the next available position in a clock-wise direction; however, if all positions are taken then the algorithm resolves this by setting the fitness to zero.\n\nThe Java-based DSGA and GA used here have been reliably run on a 1.86 GHz processor with 4 GB of memory (i.e. a standard MacBook Air). Minimal system requirements are a functional system that is able to support Java. Sample input data can be found in Supplementary File 1 but are basically an amino acid sequence with amino acids translated into hydrophilic (P) and hydrophobic (H) and the parameters for the DSGA. Sample output data are also provided (Supplementary File 2) but is basically a list of the best individuals with their corresponding calculated Free Energy value. All positive free energy values in the output should be interpreted as negative values, and vice versa. For example, a free energy value of ‘8’ for an individual in the output should be interpreted as ‘-8’. This is quite important as negative free energy values indicate spontaneous folding and positive values indicate that energy needs to be added to the system to get the protein to fold.\n\n\nResults\n\nTo demonstrate that de novo protein folding is better addressed by DSGA, we have used two different methods. Two simple proteins and a method to model the protein folding were selected. All possible combinations of the folding were computed to demonstrate that there are multiple optima. Second a traditional GA was compared to DSGA to solve for a 20 amino acid protein.\n\nWe selected a simple protein of four residues with the following sequence of hydrophilic (P) and hydrophobic (H) residues: HPHPP. The individual, which best represents how to fold the protein is 0121 (Figure 1). The first amino acid is placed in the center with the second one above (0121) the third to the right (0121), the fourth below (0121) and the last to the right (0121).\n\nHydrophilic (P) residues are represented as non-shaded squares and hydrophobic (H) residues are shown as shaded squares.\n\nNext, we moved to a more complex protein with ten amino acids, which translated into the following sequence of H and P residues: HPPHPPHPPH, and determined all the possible ways it could fold using the model described above. Since it has 10 amino acids, all the individuals generated by our DSGA will have nine genes. The values for each range from 000000000 (complete set of amino acids one above the other) to 333333333 (complete set of amino acids each one to the left of the other). Since there are four directions to place the next amino acids, there are 49 or 262,144 different ways to fold the amino acids.\n\nFigure 2 shows a graph of all of the different ways to fold HPPHPPHPPH. The X-axis contains the different ways to fold the protein. The Y-axis is the free energy for the folding method. With this folding method there are eight global optima that each have a value of four and local optima with values of three, two or one. The optimal folding is seen in Figure 3.\n\nLeft side of X-axis corresponds to 000000000 while the right side corresponds to 333333333.\n\nHydrophilic (P) residues are represented as non-shaded squares and hydrophobic (H) residues are shown as shaded squares.\n\nNext, we executed our DSGA and a traditional GA26 (Table 1) at solving the folding for the following: HPHPPHHPHPPHPHHPPHPH. This protein of 20 amino acids was used previously27,28,29 and found to have an optimal folding pattern that resulted in a free energy of -9. Since DSGA usually takes longer to run than a traditional GA, two sets of results were created for the traditional GA. One contained the same number of generations as DSGA. The other had additional generations to get the total run-time the same between the two algorithms.\n\nThe parameters between DSGA and the traditional GA were kept consistent when possible; however, DSGA does have some additional parameters not used in traditional GAs (Table 2). The first set of results was from the traditional GA running the same number of generations as DSGA. The second set of results was the traditional GA running for the same amount of time as DSGA. In this example, a traditional GA running for 6,000 generations takes about the same amount of time as DSGA running for 1,000 generations. Table 3 shows the best individuals produced for each algorithm in 15 trials. In the case of DSGA the optimum is the best individual on the Tabu List. For the traditional GA the optimum is the best individual in the last generation.\n\n\nConclusion\n\nHere we showed that even employing a simple modeling method for protein folding results in the generation of multiple local and global optima. The above also shows that using the DSGA, which is specialized for multiple optima domains, produces better results than a traditional GA. This should be instructive to researchers working on de novo techniques as many algorithms applied to protein folding are actually hybrid applications that use a GA27–29. It is possible that previous poor results could be caused by the GA’s weakness of finding local optima. Using an NGA in these algorithms could overcome this and produce improved results.\n\n\nSoftware availability\n\nZenodo: DSGA and GA from ‘Niche Genetic Algorithms are better than traditional Genetic Algorithms for de novo Protein Folding’ doi: 10.5281/zenodo.1190230\n\nMIT License.", "appendix": "Author contributions\n\n\n\nMB and JAC conceived the study and designed the experiments. MB and TB did the coding of the DSGA. MB performed the experiments. MB and JAC analyzed the data. JAC, MB, and TB wrote the paper.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors would like to thank UMUC and all the members of the ITS Department for providing a positive space to perform our research.\n\n\n\n\nSupplementary File 1: Sample input data. This file contains a sample amino acid sequence (input data) and the meanings of each parameter that needs to be set.\n\nDynamic-radius Species-conserving Genetic Algorithm for de novo Protein Folding Instructions.\n\nDynamic-radius Species-conserving Genetic Algorithm for de novo Protein Folding is a Java application written in Java version 1.7, but it should run on other versions of Java.\n\nThere are 10 command line parameters that should be set:\n\n# position 1 - population size\n\n# position 2 - number of generations\n\n# position 3 - mutation rate (decimal)\n\n# position 4 - initial radius (decimal)\n\n# position 5 - radius delta (decimal)\n\n# position 6 - reevaluation loop count\n\n# position 7 - convergence limit\n\n# position 8 - protein\n\n# position 9 - output file location\n\n# position 10 - log status 0 few logs; 1 more logs; 2 most logs\n\nAn output file path must be placed in the file location for position 9.\n\nHere is an example for running the application:\n\njava -jar /Users/mbrown15/NetBeansProjects/DSGAProteinFolding/dist/DSGAProteinFoldingKG.jar 1000 2000 0.03 8.0 -1.0 500 4 HPPHPPHPPH //Users//mbrown15//Documents//genetic-algorithm-files// 1\n\nThis method allows multiple runs to be placed in a batch file, which can be set up as follows:\n\njava -jar /Users/mbrown15/NetBeansProjects/DSGAProteinFolding/dist/DSGAProteinFoldingKG.jar 1000 2000 0.03 8.0 -1.0 500 4 HPPHPPHPPH //Users//mbrown15//Documents//genetic-algorithm-files// 1\n\n1000 12000 0.03 8.0 -1.0 500 4 HPPHPPHPPH //Users//mbrown15//Documents//genetic-algorithm-files// 1\n\n1000 20000 0.03 8.0 -1.0 500 4 HPPHPPHPPH //Users//mbrown15//Documents//genetic-algorithm-files// 1\n\nSupplementary File 2: Sample output data. This file contains a sample of the output data.\n\nTABU LIST\n\nIndividual 032321213 natural fittness 4.0\n\nIndividual 032320213 natural fittness 4.0\n\nIndividual 032320203 natural fittness 4.0\n\nIndividual 210012230 natural fittness 3.0\n\nIndividual 210212230 natural fittness 3.0\n\nIndividual 200212230 natural fittness 3.0\n\nIndividual 210212200 natural fittness 3.0\n\nIndividual 200212200 natural fittness 3.0\n\nIndividual 123320123 natural fittness 3.0\n\nIndividual 123310123 natural fittness 3.0\n\nIndividual 031321013 natural fittness 3.0\n\nIndividual 301133022 natural fittness 3.0\n\nIndividual 301133021 natural fittness 3.0\n\nBest individual(s)\n\nIndividual = 032321213 4.0\n\nIndividual = 032320213 4.0\n\nIndividual = 032320203 4.0\n\nSample output data above are the result from the following input:\n\nPopulation Size: 50\n\nNumber of generations: 5000\n\nMutation rate: 0.003\n\nInitial radius: 12.0\n\nRadius delta: -2.0\n\nReevaluation loop counter: 1000\n\nConvergence limit: 3\n\nProtein: HPPHPPHPPH\n\nThe output of the program is the full Tabu List and the best individuals for that run. The best individuals are determined by an individual’s free energy value. Free energy values are the last number in each row of the output data. So for the above sample output all three best individuals have a free energy value of -4.\n\nNOTE: All positive free energy values in the output should be interpreted as negative values, and vice versa. For example, the above free energy value of ‘4’ for the best individuals should be interpreted as ‘-4’. This is quite important as negative free energy values indicate spontaneous folding and positive values indicate that energy needs to be added to the system to get the protein to fold.\n\n\nReferences\n\nTeich M, Needham DM: in A Documentary History of Biochemistry, 1770–1940. (Rutherford, NJ : Fairleigh Dickinson University Press). 1992. Reference Source\n\nKhoury GA, Smadbeck J, Kieslich CA, et al.: Protein folding and de novo protein design for biotechnological applications. Trends Biotechnol. 2014; 32(2): 99–109. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDill KA, MacCallum JL: The protein-folding problem, 50 years on. Science. 2012; 338(6110): 1042–1046. PubMed Abstract \| Publisher Full Text\n\nFoster DP, Pinettes C: Statistical mechanics of the two-dimensional hydrogen-bonding self-avoiding walk including solvent effects. Phys Rev E Stat Nonlin Soft Matter Phys. 2008; 77(2 Pt 1): 021115. PubMed Abstract \| Publisher Full Text\n\nUnger R, Moult J: Genetic algorithms for protein folding simulations. J Mol Bio. 1993; 231(1): 75–81. PubMed Abstract \| Publisher Full Text\n\nAlberts B, Johnson A, Lewis J, et al.: Molecular Biology of the Cell, Fifth Edition. (Garland Science). 2007. Reference Source\n\nJung J, Han KY, Koh HR, et al.: Effect of single-base mutation on activity and folding of 10–23 deoxyribozyme studied by three-color single-molecule ALEX FRET. J Phys Chem B. 2012; 116(9): 3007–12. PubMed Abstract \| Publisher Full Text\n\nCoontz R, Fahrekamp-Uppenbrink J, Lavine M, et al.: Going from Strength to Strength. Science. 2014; 343(6175): 1091. PubMed Abstract \| Publisher Full Text\n\nMarion D: An introduction to biological NMR spectroscopy. Mol Cell Proteomics. 2013; 12(11): 3006–25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHenen MA, Coudevylle N, Geist L, et al.: Toward rational fragment-based lead design without 3D structures. J Med Chem. 2012; 55(17): 7909–19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPantazes RJ, Grisewood MJ, Maranas CD: Recent advances in computational protein design. Curr Opin Struct Biol. 2011; 21(4): 467–72. PubMed Abstract \| Publisher Full Text\n\nDurand P, Lehn P, Callebaut I, et al.: Active-site motifs of lysosomal acid hydrolases: invariant features of clan GH-A glycosyl hydrolases deduced from hydrophobic cluster analysis. Glycobiology. 1997; 7(2): 277–84. PubMed Abstract \| Publisher Full Text\n\nAnfinsen CB: Principles that govern the folding of protein chains. Science. 1973; 181(4096): 223–230. PubMed Abstract \| Publisher Full Text\n\nMartí-Renom MA, Stuart AC, Fiser A, et al.: Comparative protein structure modeling of genes and genomes. Annu Rev Biophys Biomol Struct. 2000; 29: 291–325. PubMed Abstract \| Publisher Full Text\n\nKaczanowski S, Zielenkiewicz P: Why similar protein sequences encode similar three-dimensional structures? Theor Chem Acc. 2009; 125(3–6): 643–50. Publisher Full Text\n\nMitchell M: in An Introduction to Genetic Algorithms. (Cambridge, MA: MIT Press). 1996. Reference Source\n\nWang C, Lefkowitz EJ: Genomic multiple sequence alignments: refinement using a genetic algorithm. BMC Bioinformatics. 2005; 6: 200. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAhn N, Park S: Finding an upper bound for the number of contacts in hydrophobic-hydrophilic protein structure prediction model. J Comput Biol. 17(4): 647–56. PubMed Abstract \| Publisher Full Text\n\nHolland JH: in Adaptation in Natural and Artificial Systems. (Ann Arbor, MI: University of Michigan Press). 1975. Reference Source\n\nBrown MS: A Species-Conserving Genetic Algorithm for Multimodal Optimization (Doctoral dissertation). Available from Dissertations and Theses database. (UMI No. 3433233). 2010. Reference Source\n\nBrown MS, Pelsoi MJ, Dirska H: Dynamic-Radius Species-Conserving Genetic Algorithm for the Financial Forecasting of Dow Jones Index Stocks. Machine Learning and Data Mining in Pattern Recognition. Ed. P. Perner (Berlin: Springer). 2013; 7988: 27–41. Publisher Full Text\n\nDe Jong KA: An analysis of the behavior of a class of genetic adaptive systems. (Doctoral dissertation, University of Michigan). Diss Abstr Int. 1975; 36(10): 5140B. (University Microfilms No. 76–9381). Reference Source\n\nLing Q, Wa G, Yang Z, et al.: Crowding clustering genetic algorithm for multimodal function optimization. Appl Soft Comput. 2008; 8(1): 88–95. Publisher Full Text\n\nLi JP, Balazs ME, Parks GT, et al.: A species conserving genetic algorithm for multimodal function optimization. Evol Comput. 2002; 10(3): 207–234. PubMed Abstract \| Publisher Full Text\n\nGlover F: Tabu Search – Part I. ORSA Journal on Computing. 1989; 1(3): 190–206. Publisher Full Text\n\nBremermann HJ: The Evolution of Intelligence: The Nervous System as a Model of its Environment. (Technical Report, No.1, Contract No. 477, Issue 17). Seattle WA: Department of Mathematics, University of Washington. 1958. Reference Source\n\nHuang C, Yang X, He Z: Protein folding simulations of 2D HP model by the genetic algorithm based on optimal secondary structures. Comput Biol Chem. 2010; 34(3): 137–142. PubMed Abstract \| Publisher Full Text\n\nSu SC, Lin CJ, Ting CK: An effective hybrid of hill climbing and genetic algorithm for 2D triangular protein structure prediction. Proteome Sci. 2011; 9(Suppl 1): S19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJiang T, Cui Q, Shi G, et al.: Protein folding simulations of the hydrophobic-hydrophilic model by combing tabu search with genetic algorithms. J Chem Phys. 2003; 119(8) 4592–4596. Publisher Full Text\n\nBrown M, Bennett T, Coker JA: DSGA and GA from ‘Niche Genetic Algorithms are better than traditional Genetic Algorithms for de novo Protein Folding’ Zenodo. 2014. Data Source" }	[ { "id": "8529", "date": "30 Apr 2015", "name": "Nathan Alexander", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors attempt to demonstrate niche genetic algorithms outperform traditional GA at de novo protein folding. They select a specific NGA, termed DSGA, to compare to traditional GA. They simplify the task of de novo protein folding into a two-dimensional problem and model amino acid interactions as hydrophilic or hydrophobic. The protein energetics use a binary approximation where the “energy” of the protein is improved by 1.0 for every pair of residues that are hydrophobic and ”adjacent but not neighbor amino acids”. Amino acid placements are limited to two-dimensional grid coordinates.The authors demonstrate that their model of protein folding can produce local optima using one ten-amino-acid sequence.The authors next use a sequence of twenty amino acids to test whether DSGA can produce a better conformation, according to their fitness function, than a traditional GA. The authors present the results of fifteen independent optimization trajectories for DSGA, traditional GA, and traditional GA that is allowed to run for additional generations. This is important because DSGA is more computationally demanding, so there would be no advantage to DSGA over traditional GA, if GA could accomplish equivalent performance to DSGA just by running additional generations to make up for the increased computational time of DSGA. The results show that DSGA is able to find the globally optimal conformation for all fifteen optimization trials, while traditional GA and traditional GA with additional generations find the optimal conformation in none of the fifteen trials.It is unclear until examination of Figure 1 what configurations on the grid are considered “adjacent” (i.e. are amino acids diagonally placed to one another considered adjacent?). The authors refer to their fitness function as calculating the free energy of the protein. However, they give no data of how their fitness function quantitatively relates to free energy. Therefore, the authors must not refer to “free energy”, but should use terms such as “fitness function” or “score”. This would also remove the discrepancy that the authors refer to free energy, but large positive values resulting from their method indicate success. The authors include twice the explanation that positive values resulting from their method should be interpreted as negative values of free energy, and negative values resulting from their method should be interpreted as positive values of free energy. This explanation will be unnecessary and it will be easier for the reader to understand the results, which provides readability incentive in addition to the scientific necessity for the authors to remove the use of the term “free energy”. As this is submitted as a Software Tool Article, in the Materials and Methods section, the authors should give explanations and any related references for what is happening in steps six through thirteen in their implementation of the DSGA pseudo-code in Table 1. The authors should provide a reference for the basis of their traditional GA implementation. In the Results section, the purpose of Figure 1 and the purpose of the description of the four amino acid protein are unclear. Does it demonstrate that with only four amino acids there already exist multiple optima? If so, a plot similar to Figure 2 should be shown. Is it just to give a simple demonstrative example of the model for protein folding? If so, it should not fall under the heading of “Multioptimum problem”. The title for Figure 2 should be revised because the use of the word “methods” is ambiguous. In the Conclusions section, the authors claim that “…DSGA, which is specialized for multiple optima domains, produces better results than a traditional GA”. This statement must be qualified to fall within the scope of their study: using their model of protein folding, DSGA “produces better results than a traditional GA” on one twenty-amino-acid sequence. The authors must specify what they mean when they say “better results”. Perhaps, they mean “configurations with higher fitness function values”. Within the constraints of their model for protein folding, the scope of the study could be broadened by testing amino acid sequences with shorter and larger length and differing hydrophobic and hydrophilic composition. This could for example demonstrate at what length is traditional GA successful and at what length does DSGA fail.Similarly, the results and conclusions summarized in the last paragraph of the Introduction must be revised to fall within the scope of the experiments. Specifically: “Here we show that protein folding is a multi-optima problem…” The authors need to change the wording to reflect the scope of their study. The authors only show that a simple model of protein folding produces a multi-optima problem. The authors could state that protein folding is a multi-optima problem and provide a reference, and then state that they show a simple model recapitulates the multi-optima nature of the problem. “…and as a result NGAs are better suited for a solution.” Is this the authors’ hypothesis? The authors should clarify their hypothesis and make it clear when they are stating their hypothesis. Are the authors attempting to show NGAs are “better suited” due to the problem having multiple optima? Are they only trying to show NGAs are “better suited”? The use of the phrase “…as a result…” suggests the former. However, the experiments performed suggest the latter ‑ that the authors are hypothesizing only about the performance of DSGA versus traditional GA. The experiments and results don’t provide any evidence as to what could be the reason for the performance difference. The authors perform no experiments to test whether the improved performance is due to an ability to overcome multi-optima ‑ such as a control experiment utilizing a single-optimum problem or sequence. When clarifying their hypothesis, the authors must specify in relation to what are NGAs better than. The hypothesis should not refer to the general class of NGAs, when only a single specific type, DSGA, is tested. Further, the authors need to define how they quantitatively measure “better suited”. Lastly, the authors need to define what they mean by “solution”. “(1) the DSGA is very adept at predicting the folded state of proteins, which was shown by selecting a 20 amino acid protein and modeling all the folds and all possible combinations;” The results do not provide evidence to support this conclusion. The results cannot show DSGA is “very adept” because the authors do not specify a quantitative measure by which “very adept” is defined, so there is no way to conclude this. Additionally, the ability of DSGA to successfully optimize the conformation of a single twenty amino acid sequence cannot be extrapolated to mean that DSGA can successfully optimize the conformation of amino acid sequences in general. Further, the phrase “folded state of proteins” must be qualified to fall within the scope of the study; specifically, that the folded state of a protein in the study is represented by a hydrophilic-hydrophobic model of amino acids on a two-dimensional grid coordinate system. \"(2) the DSGA is better than a traditional GA and better able to derive the correct folding pattern of a protein.” Similarly to the statement in the Conclusion section and above, this statement must be qualified to fall within the scope of their study. The results do not support the unqualified statement that “DSGA is better than a traditional GA”. The protein folding problem presented in the paper and used to compare DSGA to traditional GA is only a single, very specific optimization problem. This single specific optimization problem cannot be used to make the general statement that “DSGA is better than traditional GA”. It is unclear what the authors are comparing DSGA to when they state that DSGA is “…better able to derive the correct folding pattern of a protein”. Also, the authors need to specify how they measure “better” and qualify that the “folding pattern of a protein” is represented by a hydrophilic-hydrophobic model of amino acids on a two-dimensional grid coordinate system. The authors must clarify that “a protein” is a specific twenty amino acid sequence. The appropriate conclusion the results support is that, using the authors’ model of protein folding and their fitness function to score conformations, DSGA produces conformations that score better than a traditional GA on a specific amino acid sequence with length of twenty amino acids.Similarly, the conclusions in the Abstract must be revised to fall within the scope of the experiments.“DSGA is very adept at predicting the folded state of proteins”. Please see comments in 7.3. above. “DSGA is better than a traditional GA in deriving the correct folding pattern of a protein”. Please see comments in 7.4. above.Lastly, the title must be revised to clarify the scope of the study:“Niche Genetic Algorithms are better than traditional Genetic Algorithms for de novo Protein Folding.” The title must indicate the extent to which de novo protein folding is simplified: using a two-dimensional grid with a hydrophobic-hydrophilic amino acid model and binary scoring scheme. The title must also indicate the comparison between niche genetic algorithms and traditional genetic algorithms was made using a single specific niche genetic algorithm. Further, the title must indicate the two algorithms were compared using a single specific twenty-amino-acid sequence.", "responses": [ { "c_id": "1397", "date": "28 May 2015", "name": "James Coker", "role": "Author Response F1000Research Advisory Board Member", "response": "We thank the reviewer for his time and review of our work. Since there is currently only one review submitted (7 months after submission) we have decided to hold off on making adjustments to the manuscript until we hear back from other reviewers. That being said we have mentioned possible revisions in our responses below and will incorporate them based on the responses from other reviewers. Below we have cut and pasted the reviewer’s comments (numbered) and our responses to each. 1. It is unclear until examination of Figure 1 what configurations on the grid are considered “adjacent” (i.e. are amino acids diagonally placed to one another considered adjacent?).The use of the term “adjacent” in HP models has been established for over 25 years. It refers to residues that are directly left, right, up, or down (not diagonal) from a particular amino acid that is not neighboring, i.e. the previous or next amino acid (n-1 or n+1). We understand that this can be confusing for a reader unfamiliar with the field. Therefore we have made a slight modification in the “Fitness Function” section, which is the first mention of adjacent amino acids. Here we mention that these terms have been defined and used previously and list two references so readers unfamiliar with the field can learn more. 2. The authors refer to their fitness function as calculating the free energy of the protein. However, they give no data of how their fitness function quantitatively relates to free energy. Therefore, the authors must not refer to “free energy”, but should use terms such as “fitness function” or “score”. This would also remove the discrepancy that the authors refer to free energy, but large positive values resulting from their method indicate success. The authors include twice the explanation that positive values resulting from their method should be interpreted as negative values of free energy, and negative values resulting from their method should be interpreted as positive values of free energy. This explanation will be unnecessary and it will be easier for the reader to understand the results, which provides readability incentive in addition to the scientific necessity for the authors to remove the use of the term “free energy”. The use of the term ‘Free Energy’ is widely accepted in this field using the conditions we have set. See reference # 26 (Huang et al.), as well as F. Liang and W. H. Wong, J.: Chem. Phys. 2001; 115: 3374, Lau and Dill: Macromolecules. 1989: 3986-3997 (just to name a few over the past 25 years). As a result, we decided to keep the term “Free Energy” in the manuscript to be consistent with previous work. We understand that the positive values resulting from the DSGA can be confusing as positive Free Energy values correspond with a lack of spontaneous folding, which is why, as the reviewer pointed out, we twice mention that the positive results from the DSGA should be interpreted as negative values. We do understand the reviewer’s concerns. Therefore we have removed the reference to the fact that the DSGA returns values of the opposite sign in the text of the manuscript and have kept the correctly reported values. We have moved the explanation of the negative/positive values from the DSGA to the two supplementary files. In this way the reading of the manuscript will be clear and for those that choose to use the program it will be clear how it functions and how to interpret the results. 3. As this is submitted as a Software Tool Article, in the Materials and Methods section, the authors should give explanations and any related references for what is happening in steps six through thirteen in their implementation of the DSGA pseudo-code in Table 1. Steps six to thirteen basically show the portions of Dynamic-radius Species-conserving Genetic Algorithm that make it separate from a genetic algorithm. These steps include: the generation of seeds, a Tabu List, and manipulating the Tabu List and generating its final version. All of these are already explained in the Materials and Methods section and well cited. Therefore it seems redundant to put this information in Table 1. As a result we have altered Table 1 to direct the reader to the relevant section of the Materials and Methods section, etc. where further explanations/references about each step can be found. 4. The authors should provide a reference for the basis of their traditional GA implementation. We are a little uncertain as to what the reviewer is referring to here. References 16, 17, and 18 are related to Genetic Algorithms and their traditional application in sequence alignment and protein structure prediction. References 16 and 27 are in the manuscript as they are two of the most commonly sited references in relation to Genetic Algorithms. 5. In the Results section, the purpose of Figure 1 and the purpose of the description of the four amino acid protein are unclear. Does it demonstrate that with only four amino acids there already exist multiple optima? If so, a plot similar to Figure 2 should be shown. Is it just to give a simple demonstrative example of the model for protein folding? If so, it should not fall under the heading of “Multioptimum problem”. The reason Figure 1 is present in the manuscript is to provide a clear and relatively simple example of the output of the DSGA. This figure was placed where it is to serve as an introduction and explanation to the Multioptimum problem. We do not contend or intend to suggest that every protein will have a multi-optima folding method, just the opposite. Clearly the protein in Figure 1 does not have multi-optima. 6. The title for Figure 2 should be revised because the use of the word “methods” is ambiguous. The title of Figure 2 has been changed to ‘Free Energy for all possible folded forms of HPPHPPHPPH’.7. In the Conclusions section, the authors claim that “…DSGA, which is specialized for multiple optima domains, produces better results than a traditional GA”. This statement must be qualified to fall within the scope of their study: using their model of protein folding, DSGA “produces better results than a traditional GA” on one twenty-amino-acid sequence. The authors must specify what they mean when they say “better results”. Perhaps, they mean “configurations with higher fitness function values”. Within the constraints of their model for protein folding, the scope of the study could be broadened by testing amino acid sequences with shorter and larger length and differing hydrophobic and hydrophilic composition. This could for example demonstrate at what length is traditional GA successful and at what length does DSGA fail.Similarly, the results and conclusions summarized in the last paragraph of the Introduction must be revised to fall within the scope of the experiments. Specifically: 1.“Here we show that protein folding is a multi-optima problem…” The authors need to change the wording to reflect the scope of their study. The authors only show that a simple model of protein folding produces a multi-optima problem. The authors could state that protein folding is a multi-optima problem and provide a reference, and then state that they show a simple model recapitulates the multi-optima nature of the problem.2.“…and as a result NGAs are better suited for a solution.” Is this the authors’ hypothesis? The authors should clarify their hypothesis and make it clear when they are stating their hypothesis. Are the authors attempting to show NGAs are “better suited” due to the problem having multiple optima? Are they only trying to show NGAs are “better suited”? The use of the phrase “…as a result…” suggests the former. However, the experiments performed suggest the latter ‑ that the authors are hypothesizing only about the performance of DSGA versus traditional GA. The experiments and results don’t provide any evidence as to what could be the reason for the performance difference. The authors perform no experiments to test whether the improved performance is due to an ability to overcome multi-optima ‑ such as a control experiment utilizing a single-optimum problem or sequence. When clarifying their hypothesis, the authors must specify in relation to what are NGAs better than. The hypothesis should not refer to the general class of NGAs, when only a single specific type, DSGA, is tested. Further, the authors need to define how they quantitatively measure “better suited”. Lastly, the authors need to define what they mean by “solution”.3. “(1) the DSGA is very adept at predicting the folded state of proteins, which was shown by selecting a 20 amino acid protein and modeling all the folds and all possible combinations;” The results do not provide evidence to support this conclusion. The results cannot show DSGA is “very adept” because the authors do not specify a quantitative measure by which “very adept” is defined, so there is no way to conclude this. Additionally, the ability of DSGA to successfully optimize the conformation of a single twenty amino acid sequence cannot be extrapolated to mean that DSGA can successfully optimize the conformation of amino acid sequences in general. Further, the phrase “folded state of proteins” must be qualified to fall within the scope of the study; specifically, that the folded state of a protein in the study is represented by a hydrophilic-hydrophobic model of amino acids on a two-dimensional grid coordinate system.4.\"(2) the DSGA is better than a traditional GA and better able to derive the correct folding pattern of a protein.” Similarly to the statement in the Conclusion section and above, this statement must be qualified to fall within the scope of their study. The results do not support the unqualified statement that “DSGA is better than a traditional GA”. The protein folding problem presented in the paper and used to compare DSGA to traditional GA is only a single, very specific optimization problem. This single specific optimization problem cannot be used to make the general statement that “DSGA is better than traditional GA”. It is unclear what the authors are comparing DSGA to when they state that DSGA is “…better able to derive the correct folding pattern of a protein”. Also, the authors need to specify how they measure “better” and qualify that the “folding pattern of a protein” is represented by a hydrophilic-hydrophobic model of amino acids on a two-dimensional grid coordinate system. The authors must clarify that “a protein” is a specific twenty amino acid sequence. The appropriate conclusion the results support is that, using the authors’ model of protein folding and their fitness function to score conformations, DSGA produces conformations that score better than a traditional GA on a specific amino acid sequence with length of twenty amino acids.We understand the reviewer’s concerns here; however, the end result of the logic in these comments is that no one can ever really know if one algorithm is better than another since there will always be a condition that is left untested since it is impossible to test every protein and every condition. Here we show that some proteins create multi-optima problems (i.e. Figure 2), and we believe that the body of knowledge from Genetic Algorithms should encourage the use of Niche Genetic Algorithms. Our paper goes one step further and shows a test case in which, a NGA does a better job of solving the protein folding problem than a traditional GA.It is well understood by the community that currently there is no algorithm that can solve the folding problem de novo for all proteins. Even the best de novo algorithms have a low overall success rate. Also 2D models are simplifications of the actual 3D models. With that being said it is common in this field of research to make statements that the review claims to be unfounded. Consider the following quotes. Huang, C., Yang, X. & He, Z. (2010). Protein folding simulations of 2D HP model by the genetic algorithm based on optimal secondary structure, Computational Biology and Chemistry 34, pp 137-142.“GAOSS would be an efficient tool for the protein structure predictions (PSP).”Su, S., Lin, C. & Ting, C. (2011). An effective hybrid of hill climbing and genetic algorithm for 2D triangular protein structure prediction, Proteome Science 9(Suppl. 1).“The simulation results show that ERS-GA and HHGA can successfully be applied to the problem of protein structure prediction. The satisfactory simulation results validate the effectiveness of the proposed algorithms”Liang, F. and Wong, W. H. (2001). Evolutionary Monte Carlo for protein folding simulations, Journal of Chemical Physics 115(7), pp. 3374-3380.“We showed that the evolutionary Monte Carlo algorithm can be effectively applied to simulations of protein folding on lattice models.” That being said we also understand the spirit of the reviewer’s comments that we have analyzed a small number of protein sequences. Since we have analyzed more sequences we have now included that information as well. We have also revised the language of the conclusion section. 8.Similarly, the conclusions in the Abstract must be revised to fall within the scope of the experiments.1.“DSGA is very adept at predicting the folded state of proteins”. Please see comments in 7.3. above.2.“DSGA is better than a traditional GA in deriving the correct folding pattern of a protein”. Please see comments in 7.4. above.Just as we have now modified the Conclusion section we have also modified the Abstract as well. 9.Lastly, the title must be revised to clarify the scope of the study:1.“Niche Genetic Algorithms are better than traditional Genetic Algorithms for de novo Protein Folding.” The title must indicate the extent to which de novo protein folding is simplified: using a two-dimensional grid with a hydrophobic-hydrophilic amino acid model and binary scoring scheme. The title must also indicate the comparison between niche genetic algorithms and traditional genetic algorithms was made using a single specific niche genetic algorithm. Further, the title must indicate the two algorithms were compared using a single specific twenty-amino-acid sequence. We are uncertain as to how all of the reviewer’s comments here can be included in a title or how it would improve the title. DSGA is a niche genetic algorithm and we have shown that it predicts the optimally folded configuration for a protein more reliably (i.e. performs better) than a traditional genetic algorithm. Since the title does not constitute an untrue statement we do not see a valid reason to change it in the reviewer’s comments. That being said we understand that the plural form of NGA might be confusing as we use one NGA in the manuscript. Therefore we have changed the title to “A Niche Genetic Algorithm is better than traditional genetic algorithms for de novo protein folding”." } ] }, { "id": "7946", "date": "03 Jun 2015", "name": "Kenneth De Jong", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMy concern with this article in its current form is two-fold:As a software tool article, the discussion seems quite dated. The field of Evolutionary Computation has moved well beyond discussions and/or demonstrations of the form XXX is better than a simple GA. In fact, almost everything is! From a software tool perspective, the key issue here is how one deals with multimodal fitness landscapes. Various forms of nicheing GAs have been developed for this purpose since the 1980s. Additionally, other approaches such as embedding internal restart mechanisms have been developed and studied. To be of interest in 2015, the authors need to compare their approach with state-of-the-art alternatives. The particular application chosen is an important one, but the discussion also seems quite dated. The bioinformatics community has moved well beyond the simple hydrophobic-hydrophilic and on-lattice models used in this paper. To offer a credible tool to this community, one again has to do so in the context of the current state of the art. See, for example, Zhang Y (2008), or Liu J et al. (2013).", "responses": [] } ]	1	https://f1000research.com/articles/3-236
https://f1000research.com/articles/3-235/v1	06 Oct 14	{ "type": "Case Report", "title": "Case Report: Bilateral diaphragmatic dysfunction due to Borrelia Burgdorferi", "authors": [ "Suhail Basunaid", "Chris van der Grinten", "Nicole Cobben", "Astrid Otte", "Roy Sprooten", "Rohde Gernot", "Chris van der Grinten", "Nicole Cobben", "Astrid Otte", "Roy Sprooten", "Rohde Gernot" ], "abstract": "Summary:In this case report we describe a rare case of bilateral diaphragmatic dysfunction due to Lyme disease.Case report:A 62-years-old male presented to the hospital because of flu-like symptoms. During initial evaluation a bilateral diaphragmatic weakness with orthopnea and nocturnal hypoventilation was observed, without a known aetiology. Bilateral diaphragmatic paralysis was confirmed by fluoroscopy with a positive sniff test. The patient was referred to our centre for chronic non-invasive nocturnal ventilation (cNPPV). Subsequent investigations revealed evidence of anti-Borrelia seroactivity in EIA-IgG and IgG-blot, suggesting a recent infection with Lyme disease, and resulted in a 4-week treatment with oral doxycycline. The symptoms of nocturnal hypoventilation were successfully improved with cNPPV. However, our patient still shows impaired diaphragmatic function but he is no longer fully dependent on nocturnal ventilatory support.\n\nConclusion:Lyme disease should be considered in the differential diagnosis of diaphragmatic dysfunction. It is a tick-borne illness caused by one of the three pathogenic species of the spirochete Borrelia burgdorferi, present in Europe. A delay in recognizing the symptoms can negatively affect the success of treatment. Non-invasive mechanical ventilation (NIV) is considered a treatment option for patients with diaphragmatic paralysis.", "keywords": [ "Lyme disease", "diaphram", "Borrelia burgdorferi", "hypoventilation" ], "content": "Introduction\n\nPatients with bilateral diaphragmatic paralysis may initially present with dyspnea, orthopnea, and as the disease progresses respiratory failure. Bilateral diaphragmatic paralysis is a severe generalized muscle weakness, however in few cases it has been observed that the diaphragm can be the only involved organ. The most common causes of bilateral diaphragmatic paralysis are damage to the phrenic nerves and generalized muscle diseases. Nocturnal ventilatory assistance may have a significant beneficial effect6. These patients show reduced ventilatory muscle strength, as measured by maximal inspiratory and trans-diaphragmatic pressures. These symptoms could improve in association with an improved functional score and decreased dyspnea under ventilatory assistance. Non-invasive positive pressure ventilation (NPPV) is the therapeutic tool of choice for symptomatic patients with bilateral diaphragmatic paralysis.\n\nThis case report describes the development of diaphragmatic paralysis in a patient with Lyme disease with the need for ventilatory support3,4.\n\nLyme disease is a tick-borne illness caused by the spirochete Borrelia burgdorferi. There are three species of the Borrelia, all of them appear in Europe, and two appear in Asia. Lyme disease has a broad spectrum of clinical manifestations and varies in severity. Regarding the clinical manifestations of Lyme disease, three phases have been described: early localized, early disseminated and late disease. Early localized disease is characterized by the appearance of the erythema migrans, with or without constitutional symptoms. The early-disseminated disease is characterized by multiple lesions, and the late disease is typically associated with intermittent or persistent arthritis involving one or a few large joints, especially the knee. Late Lyme disease may develop months to a few years after the initial infection2.\n\n\nCase report\n\nA 62-year-old male was referred to our hospital as a second opinion for further analysis of respiratory failure due to bilateral diaphragm dysfunction. He presented initially with flu-like symptoms. These consisted of low-grade fever, arthralgia in the neck and shoulders and symptoms of nocturnal hypoventilation. The symptoms started months before the actual clinical presentation and led to deterioration of the patient’s general condition.\n\nInitially there was also a skin rash at the back of his right leg due to an unnoticed tick-bite. The rash started in the form of a ring, later progressed to a size of 10 cm in diameter. At that time the patient had also developed a numbness of the left-side of his face. This gradually resolved during the next days. He complained of dyspnoea that was worse on supine position. There was no evidence of motor/sensory abnormality in the extremities. He had no headache, but he was complaining of neck and shoulder stiffness. He developed a low-grade fever (38.7°C) without shivering. Gradually, fatigue and inactivity evolved.\n\nThe patient is an otherwise healthy Caucasian carpenter. He is married and has two healthy kids. He took no medication, had stopped smoking 32 years earlier and drank 2 units alcohol per day. History of allergy developed later when he started ceftriaxon as a second choice for peripheral neuroborreliosis. He works as a volunteer for a forest preservation fund. As a hobby he liked to walk in the woods and he was not aware of any tick-bite.\n\nInitially on physical examination, the patient was hemodynamically stable and not febrile. The fundoscopic exam was normal. The neck was supple and there was no evidence of positive meningeal signs. On percussion the left lung base was higher situated than the right lung base. In upright position our patient had a breathing rate of 24 per minute and SpO2 of 97%. Lying down for 45 second caused severe shortness of breath and an increase in respiratory rate to 40 per minute. Paradoxical breathing was observed and the saturation dropped to 91%.\n\nThe chest radiographs (Figure 1a and 1b) demonstrated an elevated left hemi-diaphragm. Screening of diaphragmatic movement during fluoroscopy with sniff manoeuvres revealed a paradoxical movement of both hemi-diaphragms (Figure 2). A pulmonary function test revealed a decrease in supine vital capacity of more than 20% of predicted (Table 2). Arterial blood gases showed pH 7.40, PaCO2 4.9kPa, PaO2 7.8kPa, HCO3 24.6 mmol/l, base excess -0.2 mmol/l. Antibodies to extractable nuclear antigens SSA, SSB, RNP, Sm, SCL-70, Jo-1 and serology of Q-fever were negative. IgG antibodies to B. burgdorferi were detectable in serum.\n\n(a) Frontal chest radiograph during initial presentation. (b) Lateral chest radiograph during initial presentation.\n\nFEV1: Forced expiratory volume in 1 second\n\nFVC: Forced vital capacity\n\nPEF: Expiratory peak flow\n\nPIF: Peak inspiratory flow\n\nFRC: Functional residual capacity\n\nRV: Residual volume\n\nTLC: Total lung capacity\n\nUltrasonography showed lack of thickening of the diaphragm with inspiration indicating a non-functioning diaphragm. Polysomnography without ventilatory support showed periods of nocturnal desaturations together with out-of-phase thoracic and abdominal movement (Figure 3 and Figure 4).\n\nFrom the nasal pressure signal (trace 5) it can be seen that breathing movements follow the inspiration.\n\nAbdominal and thoracic movements are not completely in-phase because the ventilatory support is not triggered before there is inspiratory flow.\n\nThere was no clinical evidence of central neurological abnormalities. The electromyogram (EMG) of the diaphragm revealed a normal distal motor latency with normal CMAP-amplitude of phrenic nerve on both sides. Needle EMG revealed good recruitment without spontaneous muscle activity in the right hemi-diaphragm. Technically measurement of the hemi-diaphragm was less reproducible. In conclusion there was no evidence for traumatic phrenic nerve palsy.\n\nAn extended differential diagnosis of bilateral diaphragmatic paralysis is presented below in Table 1.\n\nThe diagnosis of Lyme disease was made on the basis of serological tests demonstrating recent infection with B. burgdorferi. The diagnosis of bilateral diaphragmatic weakness was made on the basis of fluoroscopy with a sniff test (Figure 2) and ultrasonography of the diaphragm. The patient received oral doxycycline (200 mg q.d. for 4 weeks) and nocturnal support with NIV/BiPAP was started. Following therapy, our patient showed a dramatic improvement. He stopped using the nocturnal support of mechanical ventilation. He can now lie down in supine position without being orthopneic. The Epworth Sleepiness Scale (ESS) is obviously improved, and he has no other complaints. The repeated pulmonary function test showed improvement in the forced vital capacity (FVC) in supine position (from 31.5% to 65% predicted), however the difference between supine and upright position remain above the 20%. The pressure of the main inspiratory muscle is also improved in the follow-up. In the repeated polysomnography without ventilator support there was still dominant out-phase motion between abdomen and chest, which indicate persistent diaphragm dysfunction.\n\n\nDiscussion\n\nIn our case the diagnosis was based on the clinical signs and symptoms, chest radiographs and serology indicating recent infection by B. burgdorferi. Our patient was not aware of a tick-bite one year before the initial presentation, but the numbness in the left side of his face and the skin erythema spontaneously resolved within a couple of weeks, put us on track. By definition, the nervous system involvement only occurs in the disseminated phase of the infection2.\n\nThe symptoms of neurologic involvement may occur weeks to several months after tick bite and may be the first manifestation of Lyme disease1. Neurological evaluation revealed no abnormalities in our patient. Although the facial nerve is the most commonly affected cranial nerve, the classic manifestations of acute neurologic abnormalities due to Lyme disease are meningitis, cranial neuropathy, and motor or sensory radiculoneuropathy. Each of these findings may also occur individually2. Ventilatory support is very useful in acute respiratory impairment due to diaphragmatic weakness in a patient with Lyme disease.\n\nIn a case report of three patients with neuroborreliosis presenting with acute respiratory impairment, all patients presented respiratory failure associated with progressive nocturnal hypoventilation or prolonged central apnoea. Tracheostomy and prolonged periods of ventilatory support were necessary in all three cases. These cases emphasise that Borrelia infection should be considered in the differential diagnosis of unexplained respiratory failure7,9,10. Bilateral diaphragmatic paralysis is a common cause of complete respiratory failure and the symptoms could be severe4,5.\n\nIn the literature only sporadic case reports comment on the respiratory failure due to Lyme disease3.\n\nIn these cases, patients with respiratory failure caused by diaphragmatic paralysis due to Lyme disease were ventilated maximum for up to 2 months.\n\nOur patient is clinically completely recovered, but he remains, despite improvement, respiratory insufficient according to the pulmonary function test, the polysomnography and the measurement of maximal inspiratory pressure. He shows a good acceptance of the nocturnal ventilatory support. We expect a successful recovery from the phrenic nerve palsy gradually in the next 2 to 3 years. In a group of 50 patients suffering of phrenic nerve palsy about 1/3 fully recovered, 1/3 recovered in 2–4 years and the rest showed no progress in recovering9.\n\nIn conclusion, Lyme disease is an important differential diagnosis in patients with diaphragmatic paralysis. There can be an important delay between the tick bite and the development of symptoms, which has to be taken into account when dealing with these patients.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the patient.", "appendix": "Author contributions\n\n\n\nSuhail Basunaid: corresponding author, literature search and data collection. Chris van der Grinten: physiologist, head of pulmonary function department. Nicole Cobben: Chest physician, Director of the Centre for Home Mechanical Ventilation. Astrid Otte: Chest physician. Roy Sprooten: Chest Physician. Follow-up data collection. Gernot Rohde: Chest Physician, data control.\n\n\nCompeting interests\n\n\n\nThe abstract describing this work has been presented at the European Respiratory Society Annual Congress 2013.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nAbbott RA, Hammans S, Margarson M, et al.: Diaphragmatic paralysis and respiratory failure as a complication of Lyme disease. J Neurol Neurosurg Psychiatry. 2005; 76(9): 1306–1307. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCenters for Diseases Control and Prevention (CDC). Lyme disease--United States, 2003–2005. MMWR Morb Mortal Wkly Rep. 2007; 56(23): 573–6. PubMed Abstract\n\nDavis J, Goldman M, Loh L, et al.: Diaphragm function and alveolar hypoventilation. Q J Med. 1976; 45(177): 87–100. PubMed Abstract\n\nMcCool FD, Tzelepis GE: Dysfunction of the diaphragm. N Engl J Med. 2012; 366(10): 932–942. PubMed Abstract \| Publisher Full Text\n\nGibson GJ: Diaphragmatic paresis: pathophysiology, clinical features, and investigation. Thorax. 1989; 44(11): 960–70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGomez de la Torre R, Suarez del Villar R, Alvarez Carreno F, et al.: [Diaphragmatic paralysis and arthromyalgia caused by Lyme disease]. An Med Interna. 2003; 20(1): 47–9. PubMed Abstract\n\nHalperin JJ: Lyme disease and the peripheral nervous system. Muscle Nerve. 2003; 28(2): 133–43. PubMed Abstract \| Publisher Full Text\n\nMelet M, Gerard A, Voiriot P, et al.: [Fatal meningoradiculoneuritis in Lyme disease]. Presse Med. 1986; 15(41): 2075. PubMed Abstract\n\nMulvey DA, Aquilina RJ, Elliott MW, et al.: Diaphragmatic dysfunction in neuralgic amyotrophy: an electrophysiologic evaluation of 16 patients presenting with dyspnea. Am Rev Respir Dis. 1993; 147(1): 66–71. PubMed Abstract \| Publisher Full Text\n\nSilva MT, Sophar M, Howard RS, et al.: Neuroborreliosis as a cause of respiratory failure. J Neurol. 1995; 242(9): 604–7. PubMed Abstract \| Publisher Full Text\n\nSigler S, Kershaw P, Scheuch R, et al.: Respiratory failure due to Lyme meningoradiculitis. Am J Med. 1997; 103(6): 544–547. PubMed Abstract \| Publisher Full Text\n\nFaul JL, Ruoss S, Doyle RL, et al.: Diaphragmatic paralysis due to Lyme disease. Eur Respir J. 1999; 13(3): 700–702. PubMed Abstract \| Publisher Full Text\n\nWinterholler M, Erbguth FJ: Tick bite induced respiratory failure. Diaphragm palsy in Lyme disease. Intensive Care Med. 2001; 27(6): 1095. PubMed Abstract \| Publisher Full Text" }	[ { "id": "7198", "date": "05 Jan 2015", "name": "Sezai Celik", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors report a patient with bilateral diaphragmatic paralysis due to Borrelia, which improved with antibiotherapy and non-invasive ventilation. The manuscript is quite well written and sufficiently well documented. The duration of NIV/BiPAP should be clearly pointed out. İt can be accepted for indexing in its current form.", "responses": [] }, { "id": "7473", "date": "27 Jan 2015", "name": "W.N. Welvaart", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors report a patient with bilateral diaphragmatic dysfunction due to Borrelia.I enjoyed reading this interesting and well written manuscript.I think it would be nice to point out more details about the etiology of functional disorders of the diaphragm and acquired paralysis to explain why Lyme desease should be considered in the differential diagnosis of functional disorders of the diaphragm beside the mentioned extended differential diagnosis in table 1.Despite my comments, I think that in its current form it can be accepted for indexing.", "responses": [] } ]	1	https://f1000research.com/articles/3-235
https://f1000research.com/articles/3-233/v1	03 Oct 14	{ "type": "Correspondence", "title": "Activity artifacts in drug discovery and different facets of compound promiscuity", "authors": [ "Jürgen Bajorath" ], "abstract": "Compounds with apparent activity in a variety of assays might disable target proteins or produce false assay signals in the absence of specific interactions. In some instances, such effects are easy to detect, in others they are not. Observed promiscuity of compounds might be due to such non-specific assay artifacts. By contrast, promiscuity might also result from specific interactions with multiple targets. In the latter case, promiscuous compounds can be attractive candidates for certain therapeutic applications. However, compounds with artificial activity readouts are often not recognized and are further progressed, which presents a substantial problem for drug discovery. In this context, the concept of PAINS (pan-assay interference compounds) should be seriously considered, which makes it possible to eliminate flawed compounds from the discovery pipeline, even if their activities appear to be sound at a first glance.", "keywords": [ "pan-assay interference", "promiscuity", "drug discovery" ], "content": "Correspondence\n\nIn a recent commentary, Baell and Walters specify the threat to drug discovery programs that comes along with PAINS (pan-assay interference compounds)1. PAINS are small molecules that fake biological (target-specific) activities in assays, by chemically disabling target proteins or producing false assay signals (e.g., through color effects). PAINS do their fatal job through a variety of unwanted chemical mechanisms including, among others, covalent modifications, chelation of metal ions essential for catalytic functions redox effects or by disrupting membrane environments required for receptor integrity. The PAINS concept was originally introduced Baell and Holloway2 and might also be viewed in context of earlier work on frequent hitters in screening assays by the Shoichet group3. Frequent hitters cause non-specific protein aggregation, micelle formation, or denaturing effects and thereby also produce activity artifacts. The PAINS concept is knowledge-based and it is evident that a high level of chemical expertise and much careful research have been prerequisites for its introduction and further refinement.\n\nIn their off-the-beaten-path contribution, Baell and Walters detail PAINS liabilities. They provide evidence that PAINS often make it into the discovery pipeline and that their destructive deeds might be discovered late in the game, if at all, thereby wasting valuable resources. As leading experts, the authors do not hesitate to admit that they themselves have made PAIN(S)ful experiences in the past that have inspired them to dig deep and get to the roots of the problem. Indeed, PAINS often progress below the radar screen of chemical awareness and are difficult to detect when observing apparent (yet artificial) dose-response behavior and/or pseudo-SARs (structure-activity relationships).\n\nBaell and Walters point out that they have identified about 400 compound classes (!) representing PAINS, but that consideration of only 16 major classes is sufficient to eliminate more than half of PAINS present in screening libraries. Among others, these primary suspects include hydrogen peroxide producing molecules such as toxoflavins, covalent modifiers such as isothiazolones and rhodanines, or compounds whose degradation products might produce artificial signals in many assays such as phenol-sulfonamides. Baell and Walters provide guidance on how to best identify PAINS early on and prevent the progression of flawed compounds, for example, through the use of orthogonal assays to re-evaluate screening hits or computational (substructure) filters to detect PAINS. The latter approach is easy to implement and can be used on a routine basis to screen compound collections for major classes of PAINS.\n\nInterestingly, Baell and Walters attribute PAINS progression in discovery projects primarily to the naivety of medicinal chemists or drug discovery researchers in academia, a point that might be perceived as controversial by many. After all, potential PAINS pitfalls of academic scientists are easier to spot than those of their colleagues in the pharmaceutical industry because academic accidents are primarily manifested in publications, whereas failures in pharma environments are typically not publicized. On a lighter note, the discussion of Baell and Walters is supported by truly ‘innovative’ display items that should help to open up (even ugly) chemistry to the masses.\n\nFrom reviewer and editorial experiences, one can attest to the fact that PAINS present a problem for the scientific literature and often go unnoticed. For example, the Journal of Medicinal Chemistry frequently receives submissions reporting hits from experimental or computational (virtual) screening campaigns with PAINS liability (and is currently taking appropriate measures to tackle these problems, in collaboration with Jonathan Baell). Hence, there are all good reasons to raise the awareness of these issues and provide catalogues of PAINS as references for investigators in academia and the pharmaceutical industry. Of course, it is not certain that each and every compound containing a PAINS (sub)structure will be a ‘chemical con artist’ (to use Baell’s and Walters’ terminology), as compound reactivity or other effects might also be context-dependent. Any PAINS alert, however, should trigger careful follow-up studies to re-evaluate activity readouts.\n\nIn the context of PAINS and frequent hitters, another aspect should also be carefully considered, i.e., the Janus headed issue of compound promiscuity4, which is often misunderstood. Promiscuity might well be associated with non-specific effects. Compounds active in many different assays are indeed likely to represent PAINS or frequent hitters due to the artifacts discussed above. This type of compound promiscuity might be best rationalized as ‘assay promiscuity’. By contrast, promiscuity also results from the ability of small molecules to specifically interact with multiple targets4, and this type of ‘target promiscuity’ provides the molecular basis of ‘polypharmacology’5. In certain therapeutic areas such as oncology, the efficacy of a drug often depends on its ability to specifically bind to multiple targets and elicit polypharmacological effects (i.e., interference with multiple signaling pathways), with kinase inhibitors being a prime example.\n\nLarge-scale mining of assay data has revealed different facets of promiscuity4,6. Analysis of compounds from 1085 confirmatory bioassays for 439 targets available in PubChem7 has shown that a screening hit interacted with, on average, two targets, provided that only high-confidence activity data were considered6. This reflects a fairly low level of target promiscuity, although nearly 80% of all active PubChem compounds were tested in more than 50 different assays. The probability of an active compound to interact with at least two targets was calculated as ∼50% and the probability of interacting with more than five targets was less than 8%. However, even under the most stringent activity data selection criteria, more than 2000 hits (∼0.45% of PubChem’s confirmatory bioassay compound collection) were detected that were active against more than 10 targets (consistently displaying dose-response behavior)6. In these cases, boundaries between target and assay promiscuity become rather fluid, and promiscuous compounds and their activities should be further investigated. Moreover, 160 compounds displayed activities against more than 20 targets and many of these highly promiscuous PubChem compounds were PAINS, according to Baell et al.\n\nCompounds that are not PAINS or frequent hitters might occasionally also display high levels of assay promiscuity. For example, under the conditions of small molecule microarray experiments, some compounds from diversity-oriented synthesis and other sources were found to be active against more than 90 (!) sequence-unrelated targets8. Small chemical modifications of these highly promiscuous molecules (identified through matched pair analysis of library compounds) often dramatically reduced their microarray activities or rendered them completely inactive8. Hence, for such structural analogs, assay and target promiscuity would be very difficult to distinguish in the context of a given experiment, which would require follow-up assays under different conditions.\n\nThe PAINS concept put forward by Baell et al. is a milestone event for medicinal chemistry and drug discovery, just as the first detection of frequent hitters by McGovern et al. has been more than a decade ago3. Without doubt, focusing on flawed compounds represents a major obstacle for drug discovery research, be it in academia or the pharmaceutical industry, and so do publications reporting, in good faith, apparent activities of such compounds. Care must also be taken to distinguish between (true) target and assay promiscuity of active compounds and be aware of experimental situations where this might not be possible.\n\nIt has taken a fairly long time until the concept of frequent hitters was generally accepted in biological screening and it will take time until there is general awareness and routine consideration of PAINS in the practice of medicinal chemistry and drug discovery. To these ends, the commentary of Baell and Walters makes an invaluable contribution. To a wider audience (hopefully including many students) it also demonstrates that serious problems in chemistry can be dealt with in an equally thought-provoking and entertaining manner.", "appendix": "Author contributions\n\n\n\nJB selected the articles for correspondence, reviewed the data, and prepared the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBaell J, Walters MA: Chemistry: Chemical con artists foil drug discovery. Nature. 2014; 513(7519): 481–483. PubMed Abstract \| Publisher Full Text\n\nBaell JB, Holloway GA: New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J Med Chem. 2010; 53(7): 2719–2740. PubMed Abstract \| Publisher Full Text\n\nMcGovern SL, Caselli E, Grigorieff N, et al.: A common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening. J Med Chem. 2002; 45(8): 1712–1722. PubMed Abstract \| Publisher Full Text\n\nHu Y, Bajorath J: High-resolution view of compound promiscuity. [v2; ref status: indexed, http://f1000r.es/1ig]. F1000Res. 2013; 2: 144. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaolini GV, Shapland RH, van Hoorn WP, et al.: Global mapping of pharmacological space. Nat Biotechnol. 2006; 24(7): 805–815. PubMed Abstract \| Publisher Full Text\n\nHu Y, Bajorath J: What is the likelihood of an active compound to be promiscuous? Systematic assessment of compound promiscuity on the basis of PubChem confirmatory bioassay data. AAPS J. 2013; 15(3): 808–815. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang Y, Xiao J, Suzek TO, et al.: PubChem’s BioAssay Database. Nucleic Acids Res. 2012; 40(Database issue): D400–D412. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDimova D, Hu Y, Bajorath J: Matched molecular pair analysis of small molecule microarray data identified promiscuity cliffs and identifies molecular origins of extreme compound promiscuity. J Med Chem. 2012; 55(22): 10220–10228. PubMed Abstract \| Publisher Full Text" }	[ { "id": "6317", "date": "06 Oct 2014", "name": "John A. Lowe III", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors make a good case for the importance of the recent publication on PAINS compounds, hits from screening that are artifacts and may drain resources in followup form legitimate hits. Their conclusion that it will take years for the scientific community to accept this case and institute controls to protect against it is also valid. They point some caveats in terms of compounds that are legitimately promiscuous and therapeutically valuable as a result. But for academic labs unfamiliar with the HTS triage process, the danger of wasting resource on PAINS will be an ongoing risk of the drug discovery process. The article thus makes many valid and worthwhile points, and should be indexed.", "responses": [] }, { "id": "6319", "date": "09 Oct 2014", "name": "Bill Greenlee", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting and informative paper on the concept of PAINS (pan-assay interference compounds), compounds that have apparent activity in multiple assays, but in fact produce false signals due to non-specific interactions or interference with the assays. As indicated by Baell and Walters (2014) in a recent commentary, PAINS can lead to confusion and wasted resources. In this paper, Bajorath points out that promiscuity, one of the characteristics of PAINS, can also be due to the ability of small molecules to specifically interact with multiple targets, which provides the molecular basis of ‘polypharmacology.' This paper highlights the difficulties that may be encountered in distinguishing between these two possibilities.", "responses": [] }, { "id": "6316", "date": "21 Oct 2014", "name": "Peter R Bernstein", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article provides interesting commentary on pan-assay interference compounds [PAINS] and focuses on the recent paper by Baell and Walters (2014). Because of the danger of wasting resources following up inappropriate leads, major effort has gone into identifying classes of compounds that are made up of PAINS. One danger of applying many PAINS filters is that they will remove compounds that are promiscuous but are real leads, as highlighted by the authors reference to the work of Dimova and Bajorath (2012). There is a delicate balance between removing PAINS and keeping real, small \"promsicuous\" hitters. In addition to that reference I highlight the paper of Skolnick and Gao (2013) which explains the basis for promiscuous interactions with proteins and provides a potential paradigm for identifying promiscuous compounds that are not PAINS.", "responses": [] } ]	1	https://f1000research.com/articles/3-233
https://f1000research.com/articles/3-231/v1	02 Oct 14	{ "type": "Research Article", "title": "Characterization of population-based variation and putative functional elements for the multiple-cancer susceptibility loci at 5p15.33", "authors": [ "Lisa Mirabello", "Charles C. Chung", "Meredith Yeager", "Sharon A Savage", "Charles C. Chung", "Meredith Yeager", "Sharon A Savage" ], "abstract": "Background:TERT encodes the telomerase reverse transcriptase, which is responsible for maintaining telomere ends by addition of (TTAGGG)n nucleotide repeats at the telomere. Recent genome-wide association studies have found common genetic variants at the TERT-CLPTM1L locus (5p15.33) associated with an increased risk of several cancers. Results:Data were acquired for 1627 variants in 1092 unrelated individuals from 14 populations within the 1000 Genomes Project. We assessed the population genetics of the 5p15.33 region, including recombination hotspots, diversity, heterozygosity, differentiation among populations, and potential functional impacts. There were significantly lower polymorphism rates, divergence, and heterozygosity for the coding variants, particularly for non-synonymous sites, compared with non-coding and silent changes. Many of the cancer-associated SNPs had differing genotype frequencies among ancestral groups and were associated with potential regulatory changes. Conclusions:Surrogate SNPs in linkage disequilibrium with the majority of cancer-associated SNPs were functional variants with a likely role in regulation of TERT and/or CLPTM1L. Our findings highlight several SNPs that future studies should prioritize for evaluation of functional consequences.", "keywords": [ "TERT", "CLPTM1L", "population genetics", "5p15.33" ], "content": "Introduction\n\nThe 5p15.33 locus includes the TERT (human telomerase reverse transcriptase) and the CLPTM1L (alias CRR9; cleft lip and palate transmembrane 1 like) genes. Telomerase reverse transcriptase (TERT) is the essential catalytic component of the telomerase holoenzyme responsible for maintaining telomere ends. Telomerase compensates for DNA polymerase’s inability to fully replicate the lagging DNA strand by adding hexanucleotide (5'-TTAGGG-3')n repeats to the 3’ end of chromosomes using a template sequence within the RNA component (TERC) of the enzyme1. Telomeres, consisting of these hexanucleotide repeats and several associated proteins, are responsible for preserving chromosomal stability by protecting chromosomes from end-to-end fusion, atypical recombination, and degradation2. In normal differentiated cells, expression of telomerase is very low or absent and telomeres erode by 50 to 200 base pairs with each cell division1. When the telomeres become critically short, they act as a cellular clock and signal cellular senescence and apoptosis3,4. In contrast, telomerase activity has been detected in 90% of human cancers5,6 and allows these malignant cells to continually divide by bypassing cellular crisis7.\n\nCLPTM1L is located approximately 23 kilobases (kb) centromeric of TERT. Little is known about the function of the CLPTM1L protein. It is a predicted transmembrane protein that is expressed in a range of normal and malignant tissues including skin, lung, breast, ovary and cervix, and has been shown to sensitize ovarian cancer cells to cisplatin-induced apoptosis8.\n\nThe clinically related telomere biology disorders (TBDs), such as pulmonary fibrosis or aplastic anemia, are associated with germline mutations causing amino acid substitutions, additions, deletions, and frame shift mutations within TERT9,10. Patients with the more severe TBD, dyskeratosis congenita (DC) have very high risks of bone marrow failure and cancer, and have telomeres below the 1st percentile for their age11. DC represents the most clinically severe outcome of germline TERT mutations and often presents in childhood. Individuals with isolated aplastic anemia or pulmonary fibrosis due to TERT mutations tend to manifest clinical symptoms in adulthood.\n\nGenome-wide association studies (GWAS) have found that common genetic variants, in the form of single nucleotide polymorphisms (SNPs), within the TERT-CLPTM1L locus (5p15.33) are associated with relatively low but highly statistically significant risks (odds ratios for risk alleles ranging between 1.05–1.6) of several cancers, including glioma12,13, basal cell carcinoma14,15, testicular16, pancreatic17, lung18–20, bladder21, colorectal22, breast23, and overall cancers24 [reviewed in25,26].\n\nBoth TERT and CLPTM1L are evolutionarily conserved across diverse species, which suggests their functional importance8,27,28. TERT has low nucleotide diversity, and common SNPs in this gene region show low levels of differentiation among populations and high ancestral allele frequencies28,29; this pattern of low overall diversity suggests that TERT may be constrained29.\n\nThe 1000 Genomes Project Consortium has reported that different populations have different profiles of rare and common variants; and, varying degrees of purifying selection at functionally relevant low-frequency sites which lead to substantial local population differentiation30. Large surveys of human genetic variation have described an excess of rare genetic variants as a result of a recent population expansion and weak purifying selection31–33, particularly for variants in disease genes and for individuals of European ancestry33.\n\nIn order to better understand the population genetics underlying the 5p13.3 locus associated with cancer, we conducted a detailed analysis of allele frequency patterns among ancestral group, levels of differentiation, and recombination at the 5p15.33 locus using 1000 Genomes Project34 data. We retrieved data for the TERT-CLPTM1L genes and flanking regions for 1092 individuals from 14 populations. Analyses were focused on understanding how allele frequencies differ between populations, and evaluation of the cancer-associated SNPs and their surrogate markers for potential functional elements.\n\n\nMaterials and methods\n\nData were retrieved for 1627 variants on 5p15.33 (hg19, chr5: 1,243,287–1,355,002) for all individuals in the 14 populations (1092 individuals) included in the 1000 Genomes project (2012 February release)34. Eighteen potentially related individuals were removed, which resulted in 1074 individuals. We also retrieved data for a flanking region, approximately 10kb upstream and downstream, in order to improve understanding of these gene regions [Data File 1].\n\nThe package ARLEQUIN version 3.535 was used to compute FST values, diversity, AMOVA, and heterozygosity. FST values based on allele frequencies were calculated as a measure of population differentiation, and significance was estimated with 10,000 permutations; and, these levels were compared to the genome-wide average for autosomal SNPs (FST ≈ 0.136–39). The population of African-Americans in the Southwestern United States (ASW) was grouped with the two populations of West African ancestry (Luhya in Kenya [LWK] and Yoruba in Nigeria [YRI]) since in our population level analyses they were found to be most closely related to these individuals of African ancestry, as previously observed40. In order to apportion the fraction of the genetic variance due to differences between and within ancestral groups (European, East Asian, West African, and American) and infer the genetic structure of the populations, AMOVA was performed with 10,000 permutations. HAPLOVIEW version 4.141 was used to determine the degree of linkage disequilibrium (LD) and minor allele frequency (MAF). The GLU genetics’ ld.tagzilla module was used for the tag analysis with a LD pairwise r2 threshold of 0.8. Pairwise LD was analyzed separately for the four ancestral groups and used to select tag SNPs for each region.\n\nSNPs within TERT and CLPTM1L were grouped by functional category (i.e., coding vs. non-coding, and synonymous vs. non-synonymous variants), and tested for significant differences in the normalized number of variant sites, allelic frequency divergence, heterozygosity, minor allele frequency (MAF), and levels of differentiation among populations; significant differences would suggest that these functional categories of loci were not affected similarly, as expected under the assumption of neutrality. The allelic frequency divergence between ancestral groups was computed using: d = 1-[(x1y1)1/2 + (x2y2)1/2], where x1 and y1 are the frequencies of the first allele and x2 and y2 are the frequencies of the second allele42. The normalized number of variant sites was calculated as: θ^ = K/Σn-1i=1 i-1L, where K is the number of variant sites, n is the number of chromosomes, and L is the total sequence length. Differences between the SNP functional categories were tested for significance with a two-tailed t-test. SIFT (Sorts Intolerant From Tolerant) and Polyphen 2 (Polymorphism Phenotyping v2) were used to predict the potential impact of an amino acid substitution43,44.\n\nTo identify recombination hotspots in this region, we used SequenceLDhot45, a program that uses the approximate marginal likelihood method46 and calculates likelihood ratio statistics at a set of possible hotspots. We used the four ancestral groups [European (EUR; n=379), East Asian (EA; n=286), American (AM; n=184), and African (AFR; n=246)] to calculate background recombination rates using PHASE v2.147,48. The likelihood ratio statistics of 12 predicts the presence of a hotspot with a false-positive rate of 1 in 3,700 independent tests.\n\nPutative functional elements were assessed using the UCSC genome browser (http://genome.ucsc.edu/), a publically available bioinformatics website, for ENCODE Regulation and Comparative Genomics tracks for all of the cancer-associated SNPs and their surrogates for each ancestral group. SNPs were considered surrogates for cancer-associated SNPs for each ancestral group if the r2 ≥0.60, the inter-marker distance ≤200kb, and the MAF ≥0.05. We assessed potential regions of open chromatin with DNase hypersensitivity; potential regulatory histone marks (H3K4Me1, H3K4Me3, H3K27Ac); protein binding sites; regulatory motifs; CpG islands; conserved mammalian microRNA regulatory binding sites; and evolutionary conservation among placental mammals using the phylop basewise conservation measurement49. Functional elements were also assessed using RegulomeDB, an integrated database that annotates SNPs with known or predicted regulatory DNA elements, including DNase hypersensitivity, transcription factor binging sites, and promoter regions that regulate transcription using data from GEO, ENCODE, and published literature50. RegulomeDB scores are a heuristic scoring system based on confidence that a variant is located in a functional region and likely results in a functional consequence, these are used to assist comparison among annotations50. Lower scores indicate increased evidence; category 2 scores are variants likely to affect binding, category 3 scores are less likely to affect binding; and 4, 5, or 6 scores are variants with minimal binding evidence.\n\n\nResults\n\nThere were 1627 variants in the TERT-CLPTM1L region among all individuals (N=1074): 167 were upstream of TERT, 563 in TERT (including UTR, intronic and exonic regions), 353 were between TERT and CLPTM1L (downstream of TERT and upstream of CLPTM1L), 412 in CLPTM1L (including UTR, intronic and exonic regions), and 132 downstream of CLPTM1L. A summary of the variation for the different functional categories of polymorphisms in TERT and CLPTM1L is given in Table 1. The majority of SNPs in TERT and CLPTM1L were in intronic regions (N=903), only 72 were exonic (49 in TERT and 18 in CLPTM1L). 46 of the exonic variants were synonymous changes (32 in TERT and 9 in CLPTM1L) and 26 were non-synonymous protein altering variants (PAV) (17 in TERT and 9 in CLPTM1L). The SNPs previously associated with cancer at 5p15.3325 are all located in the intronic regions of TERT or CLPTM1L or intergenic between these genes, except for one which is a coding synonymous SNP in TERT (rs2736098; Table 2).\n\n* includes intronic and 3' UTR SNPs; bp = base-pairs; Polys = polymorphisms; θ^ = normalized number of variant sites; Het. = heterozygosity; MAF = minor allele frequency; FST = level of differentiation among ancestral groups.\n\n† Ethnicity as reported in Mocellin et al. (2012); ‡ major allele:minor allele, and the risk allele is underlined; syn. = synonymous change; RAF = risk allele frequency; FST = level of differentiation among ancestral groups; misc. = miscellany, indicating a mix of different races; AFR = African ancestry; EUR = European ancestry; AM = American ancestry; EA = East Asian ancestry.\n\nSince there were so few coding variants in the TERT and CLPTM1L loci, we combined them for the following analyses. The normalized number of variant sites, heterozygosity, and MAFs were significantly different by functional SNP category in TERT and CLPTM1L (P values <0.01; Table 1). Specifically, the non-coding SNPs (compared with coding SNPs) and synonymous SNPs (compared with non-synonymous SNPs) had significantly higher numbers of variant sites, heterozygosity, and MAFs (Table 1). These trends were consistent in all ancestral groups (Figure 1A). The most significant differences between coding and non-coding SNPs were in African populations (non-coding average MAF 9.8% vs. coding average MAF 0.9%); and, the most significant differences between synonymous (syn.) versus non-synonymous (non-syn.) SNPs were in East Asian populations (syn. average MAF 4.8% vs. non-syn. average MAF 0.2%) (Figure 1A). There were significantly different levels of differentiation among ancestral groups for coding versus non-coding and synonymous versus non-synonymous SNPs (Figure 1B).\n\n(A.) Average minor allele frequency of the polymorphisms by functional category for each group; (B.) average level of differentiation among ancestral groups (FST) for the polymorphisms by functional category; (C.) minor allele frequency of each protein-altering variant by ancestral group, the underlined variants are predicted to be potentially deleterious with SIFT and/or Poly-Phen. ** indicates a significant difference with a P <0.01, * P <0.05. PAV = non-synonymous protein-altering variation; AFR = African ancestry; EUR = European ancestry; AM = American ancestry; EA = East Asian ancestry.\n\nAll PAVs were present at a rare or low frequency (Figure 1C). European ancestry individuals had higher MAFs for many of the PAVs in TERT and CLPTM1L, and there were significant MAF differences among ancestral groups for rs35719940, rs61748181, rs33955038, and rs113203740 (Figure 1C). Nine (53%) of the 17 PAVs observed in TERT and three (33%) of the nine PAVs observed in CLPTM1L were reported to be damaging by Polyphen and/or SIFT (two in silico approaches; underlined in Figure 1C). Most of these potentially damaging variants were only observed in one individual. However, three possibly damaging variants in TERT were observed in multiple individuals [rs34094720 (N=3), rs61748181 (N=31), rs200843534 (N=5)] (Figure 1C).\n\nA summary of the variation by ancestral group for this region is given in Table 3. There was low nucleotide diversity (average of 5.0E-4) by ancestral group and low differentiation among ancestral groups (90.4% of loci in this region had low FST <0.10; median FST = 0.005) (data not shown). The median FST among ancestral groups (AG) and within populations (WP) for SNPs located within TERT and CLPTM1L were low (AG FST = 0.0039 and 0.0040, respectively; and, WP FST = 0.0078 and 0.0091, respectively). The greatest level of pairwise differentiation was among African and East Asian ancestry populations (pairwise FST = 0.208), and among European and East Asian ancestry populations (pairwise FST = 0.104) (Figure 2 and Supplementary Figure 1). The lowest level of pairwise differentiation was among European and American ancestry populations (pairwise FST = 0.01). The MAFs and heterozygosity estimates for SNPs in this region in European and American ancestry populations were highly correlated (r2 = 0.95 and 0.965, respectively).\n\nSD = standard deviation.\n\nSummary of population genetics parameters in European (A.) and African (B.) ancestry individuals for 5p15.33. Linkage disequilibrium (LD), recombination hotspots, heterozygosity, and pairwise Fst values are shown for the cancer-associated SNPs (red dots), surrogate SNPs (blue dots), and non-surrogate SNPs (grey dots). LD pattern (see color legend) is shown for SNPs with a MAF ≥0.05. The red lines represent an extension of the location of the cancer-associated SNPs. The blue lines in the heterozygosity plot indicate the location of the recombination hotspots. For the pairwise Fst estimates, the populations are indicated in the top corner of each graph. AFR = African ancestry; EUR = European ancestry; AM = American ancestry; ASN = East Asian ancestry.\n\nThere was little to no LD in the TERT gene region but high LD was present in the CLPTM1L gene region (Figure 2 and Supplementary Figure 1). There were 4–5 main recombination hotspots in TERT and between TERT and CLPTM1L, there were no hotspots located within CLPTM1L (Supplementary Table 1). The greatest recombination was observed in individuals with African ancestry (5 recombination hotspots), and the lowest recombination in individuals with East Asian ancestry (4 recombination hotspots and lower likelihood ratio statistics) (Figure 2 and Supplementary Figure 1).\n\nTwenty-three SNPs significantly associated with cancer at 5p15.3325 were included in the analysis (Table 2). Many of the cancer associated SNPs in this region had differing allele frequencies and heterozygosity among ancestral groups and populations, and had FST values close to or greater than 0.1 (Table 2 and Supplementary Table 4). The risk allele was the rare allele at all of these SNPs, except at rs4246742 (associated with lung cancer; Table 2). Most of the cancer-associated SNPs in the CLPTM1L gene region are in regions of high LD, and therefore, have many surrogates (25–54 surrogate SNPs) with r2 ≥0.6 (Table 4 and Supplementary Table 2). In contrast, most of the SNPs in the TERT gene region are in a region of low LD and have no or few surrogates (0–5 surrogate SNPs) with r2 ≥0.6 (Table 4 and Supplementary Table 2). In East Asian ancestry individuals SNPs in the CLPTM1L gene region are particularly highly correlated, even some of the SNPs within TERT are in high LD in these individuals (i.e., rs10069690, rs2242652, and rs13167280; Supplementary Figure 1).\n\n† r2 ≥0.6, maximum inter-marker distance of 200kb and minimum MAF of 0.05;\n\nAFR = African ancestry; EUR = European ancestry; AM = American ancestry; EA = East Asian ancestry;\n\nExistence of a regulatory signature is indicated as dots (number of cell types this signature was observed, only indicated if occurring in ≥2 cell types);\n\nRegulomeDB score indicates: 4 = TF binding + DNase peak, 5 = TF binding or DNase peak, 6 = motif hit, — = no data available;\n\nHighlighted rows indicate that one or more surrogates for this SNP results in a likely functional consequence (RegulomeDB score of 2);\n\nMammal Conserv. = measurement of evolutionary placental mammal basewise conservation, the conserved sites are indicated.\n\nAll previously reported cancer-associated SNPs and all possible surrogates at r2 ≥0.6 were assessed for the presence of potential regulatory elements and evolutionary conservation among mammalian species (summarized in Table 4 and Supplementary Table 3). Surprisingly, none of the cancer-associated SNP surrogates were located in the coding regions of TERT or CLPTM1L. Many of these SNPs are associated with open chromatin (DNase hypersensitivity) and/or regulatory histone marks (H3K4Me1, H3K4Me3, H3K27Ac) in multiple cell types, alter known regulatory motifs and/or protein binding sites. One of the surrogate SNPs in the putative promoter region of TERT, rs2853669, is a conserved binding site for POLR2A, as were six other surrogate SNPs located intergenic between TERT and CLPTM1L, within the CLPTM1L gene region, and in the putative promoter region of CLPTM1L. One of the cancer-associated SNPs, rs2736098, and three surrogate SNPs in the 5’ region and putative promoter region of TERT were C>T SNPs located in the CpG island. Clusters of several surrogate SNPs located within CLPTM1L and just 3’ and 5’ of CLPTM1L were associated with many histone marks and open chromatin, and/or altered regulatory motifs and protein binding sites. None of the cancer-associated SNPs or their surrogates were associated with microRNA binding sites.\n\nWe used the RegulomeDB scoring system to compare and prioritize potential functional consequences of these SNPs. The cancer-associated SNPs in the 5’ region of TERT, most of the intergenic cancer-associated SNPs, and all the cancer-associated SNPs within CLPTM1L had surrogates with a likely functional consequence of affecting binding, indicated by a category 2 score (highlighted in Table 4 and Supplementary Table 3). None of the SNPs were identified to be associated with changes in expression of these genes.\n\n\nDiscussion\n\nData from the 1000 Genomes Project34 on 1627 variants at 5p15.33 for 1074 unrelated individuals were used to describe the population genetic patterns in this region. We evaluated differentiation among ancestral groups, allele frequency patterns, and the cancer-associated SNPs and surrogates for potential regulatory elements. We have previously shown that there is low nucleotide diversity and differentiation among populations in TERT and suggested that TERT may be constrained28,29; however, our previous population genetics study focused on telomere genes as a gene set and was limited to only four SNPs located within the TERT gene29. In this study with better coverage of the TERT-CLPTM1L region, we determined that there is low nucleotide diversity across the 5p15.33 region in all ancestral groups and low differentiation among groups. As expected, African populations had more diversity, specifically at non-coding SNPs, compared to the other ancestral groups. However, East Asian populations had greater diversity at synonymous SNPs, and Europeans had the greatest frequency of non-synonymous changes. European and American ancestry individuals had very similar allele frequency patterns, as others have observed51.\n\nThe significantly reduced normalized number of variant sites, heterozygosity, and MAFs, and low differentiation among ancestral groups for the coding sites, particularly for non-synonymous sites, compared with non-coding and silent changes suggests purifying selection in TERT and CLPTM1. African ancestry individuals had the greatest difference between the frequencies of non-coding vs. coding variants, consistent with stronger purifying selection; in contrast, European ancestry individuals had an excess of potentially deleterious non-synonymous SNPs. These observations are consistent with reports of genes important in cancer and complex disease42,52–54 and recent genomic reports30–33. European ancestry individuals have been reported to have an excess of recently arisen potentially deleterious variants in disease genes33. American and East Asian ancestry individuals also had an excess of coding variants compared to African ancestry individuals, suggesting weaker purifying selection in these populations as well. East Asian individuals had a particular excess of synonymous variants and very few non-synonymous variants. For the cancer-associated SNPs in this region, the risk allele was primarily the rare allele which additionally provides support for the hypothesis of constraint in this region. This evidence of purifying selection supports the importance of TERT and CLPTM1 in disease, and the variation by ancestry suggests the level of selection differs by geographic region.\n\nWe found that several of the 23 SNPs that have been significantly associated with cancer at 5p15.33 [Reviewed in 25] had differing MAFs and heterozygosity among ancestral groups. Europeans and Americans had the most similar MAFs and heterozygosity estimates, which suggests significant admixture. These differences, reflected in the high FST values, may correlate to varying disease incidence rates among ancestral groups. For example, the breast cancer associated SNP, rs1006969023, had significantly different minor allele frequencies among ancestral groups; the homozygous risk allele genotype was significantly more common in African ancestry individuals (genotype frequency of 40% vs. 2.4% in East Asian, 6.8% in American, and 8.4% in European ancestry individuals) and less common in East Asian ancestry individuals. This difference may be associated with the higher incidence of breast cancer in African ancestry individuals (particularly for estrogen receptor-negative breast cancer) and lower incidence in East Asian individuals.\n\nMany of the cancer-associated SNPs and surrogate SNPs were associated with potential regulatory elements, including histone marks, open chromatin, transcription factor binding sites, and/or regulatory motifs. There were only a few surrogates for the SNPs located within TERT and just 5’ of TERT due to the low levels of LD in these regions; and, there were a large number of surrogates for the SNPs located close to and within CLPTM1L where LD was strong and recombination low, most of these surrogates were shared among the cancer-associated SNPs in this region. Many of the surrogate markers were located in the putative promoter regions of TERT and CLPTM1L and may affect gene regulation. The RegulomeDB scoring approach allowed us to classify variants based on all of the regulatory information. This approach determined that surrogate SNPs for many of the cancer-associated SNPs are functional variants with a likely role in regulation; these should be prioritized for functional assays.\n\n\nConclusions\n\nOur analysis of diversity in this important cancer-associated region of 5p15.33 provides background information for understanding variation in the general population. The functional impact of common variation in this region needs to be examined experimentally, but we could speculate that the diversity of coding variants among different ethnicities could have mild effects on the phenotype disparity observed among these populations. Many of the cancer-associated SNPs and/or surrogates at 5p15.33 are associated with regulatory changes and candidates for evolutionary selection. Evidence of purifying selection in TERT and CLPTM1L highlights their functional importance and associations with complex disease. We have identified SNPs in this region that are likely involved in regulation of the TERT and/or CLPTM1 genes. Future studies of the functional consequences of the 5p15.33 variants will be required to understand their contribution to cancer etiology.\n\n\nData availability\n\nF1000Research: Dataset 1. Genotype data for 1627 variants on 5p15.33 (hg19, chr5: 1,243,287–1,355,002) for 1074 individuals from 14 populations, 10.5256/f1000research.5186.d3552155", "appendix": "Author contributions\n\n\n\nProject design was carried out by S.A.S., L.M., and M.Y.\n\nGenotyping data were retrieved by C.C.C.\n\nAnalyses were performed by L.M.\n\nThe manuscript was written by L.M. and S.A.S., and reviewed by all co-authors.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis project has been funded by the Intramural Research Program of the Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, and with federal funds from the National Cancer Institute, National Institutes of Health, under contract number HHSN261200800001E to M.Y. and C.C.C.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary Table 1. Recombination hotspot inference summary. Click here to access the data.\n\nSupplementary Table 2. All possible surrogate markers by ancestral group and their rank for the 23 cancer-associated SNPs based on a R2 ≥0.60, maximum inter-marker distance of 200kb, and minimum MAF of 0.05. Click here to access the data.\n\nSupplementary Table 3. Previously reported multiple-cancer susceptibility loci at 5q15.33 and their surrogates at an r2 ≥0.6 and regulatory elements. Click here to access the data.\n\nSupplementary Table 4. Risk allele frequencies of the cancer-associated SNPs at the TERT-CLPTM1L locus by population. Click here to access the data.\n\nSupplementary Figure 1. Summary of population genetics parameters in East Asian (A.) and American (B.) ancestry individuals for 5p15.33. Click here to access the data.\n\nLinkage disequilibrium (LD), recombination hotspots, heterozygosity, and pairwise Fst values are shown for the cancer-associated SNPs (red dots), surrogate SNPs (blue dots), and non-surrogate SNPs (grey dots). LD pattern (see color legend) is shown for SNPs with a MAF ≥0.05. The red lines represent an extension of the location of the cancer-associated SNPs. The blue lines in the heterozygosity plot indicate the location of the recombination hotspots. For the pairwise Fst estimates, the populations are indicated in the top corner of each graph. AFR = African ancestry; EUR = European ancestry; AM = American ancestry; ASN = East Asian ancestry.\n\n\nReferences\n\nCollins K, Mitchell JR: Telomerase in the human organism. Oncogene. 2002; 21(4): 564–579. PubMed Abstract \| Publisher Full Text\n\nMoon IK, Jarstfer MB: The human telomere and its relationship to human disease, therapy, and tissue engineering. Front Biosci. 2007; 12: 4595–4620. 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PubMed Abstract \| Publisher Full Text\n\n1000 Genomes Project Consortium, Abecasis GR, Altshuler D, et al.: A map of human genome variation from population-scale sequencing. Nature. 2010; 467(7319): 1061–1073. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCooper GM, Stone EA, Asimenos G, et al.: Distribution and intensity of constraint in mammalian genomic sequence. Genome Res. 2005; 15(7): 901–913. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoyle AP, Hong EL, Hariharan M, et al.: Annotation of functional variation in personal genomes using RegulomeDB. Genome Res. 2012; 22(9): 1790–1797. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang QY, Song J, Gibbs RA, et al.: Characterizing polymorphisms and allelic diversity of von Willebrand factor gene in the 1000 Genomes. J Thromb Haemost. 2013; 11(2): 261–269. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHughes AL, Packer B, Welch R, et al.: Widespread purifying selection at polymorphic sites in human protein-coding loci. Proc Natl Acad Sci U S A. 2003; 100(26): 15754–15757. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFreudenberg-Hua Y, Freudenberg J, Kluck N, et al.: Single nucleotide variation analysis in 65 candidate genes for CNS disorders in a representative sample of the European population. Genome Res. 2003; 13(10): 2271–2276. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHalushka MK, Fan JB, Bentley K, et al.: Patterns of single-nucleotide polymorphisms in candidate genes for blood-pressure homeostasis. Nat Genet. 1999; 22(3): 239–247. PubMed Abstract \| Publisher Full Text\n\nMirabello L, Chung CC, Yeager M, et al.: Dataset 1. Genotype data for 1627 variants on 5p15.33 (hg19, chr5: 1,243,287–1,355,002) for 1074 individuals from 14 populations. F1000Research. 2014. Data Source" }	[ { "id": "6299", "date": "10 Oct 2014", "name": "Duncan Baird", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nNumerous studies have identified variation at the TERT-CLPTM1L locus in conferring an increased risk of many different cancer types.Here the authors have examined the genetic architecture of the TERT-CLPTM1L locus using sequence data from the 1000 genomes project. Given the potential significance of this locus, this type of work is important as it has the potential to identify functional variants that might not have been uncovered with the various GWAS undertaken to identify risk variants. Thus far none of the risk variants identified at this locus with GWAS results in non-synonymous protein changes, however this study provides data to indicate that some of these variants may be associated with regulatory sequences and chromatin marks. This study also identified 26 variants that result in non-synonymous protein changes in the hTERT or the CLPTM1L genes.This is a well written manuscript and the conclusions are appropriately backed up by the data provided. The title is appropriate and the abstract adequately summarises the article. Overall this manuscript provides useful information that that will underpin future work to establish the importance of this locus in conferring cancer risk.I have no major criticisms of this work; however I recommend that a more rigorous statistical review, than I am able to provide, is undertaken of this manuscript.", "responses": [] }, { "id": "9055", "date": "16 Jun 2015", "name": "John L. Hopper", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMirabello et al present here a comprehensive bioinformatics investigation of genetic variation at the telomerase-containing locus (5p15.33) that has been associated with a range of malignancies. Given high biological plausibility of telomerase involvement in cancer pathology, this is an important study that could assist in further research on this putative susceptibility locus.The research strategy described in this well written paper should be applauded as it can be easily applied to other genomic regions of interest and provides an excellent example of extracting more useful information from existing data. In particular, the use of 1000 Genomes data provides an opportunity to examine the distribution of a wider range of variants in detail not possible using GWAS genotyping alone.As the authors point out, highly significant associations of a number of SNP variants are paralleled by rather small phenotypic associations with these variants. The most common protein altering variant (rs61748181) identified in the available data appears to have modest associations. This is not a unique situation and it makes choosing variants for functional characterization difficult considering the investment required for such comprehensive studies. It should be stressed that direct identification of causal variants from GWAS data has not been very successful. The present report demonstrates the need for well-designed analytical approach based on the sequence information (1000 Genomes) together with other data (ENCODE) to reveal credible causal candidates and narrow the choices for subsequent experimental verification. The authors acknowledge the key role of future functional work in this discovery process.As the data from 1000 Genomes Consortium comes from unaffected people inclusion of other information in the analytical pipeline that allows comparison of germline and tumour sequence information (e.g. The Cancer Genome Atlas, eQTLs) might allow further refinement of variant evaluation with different mechanisms evident in different cancers (e.g. relevance of promoter mutations - Lindner et al., 2015 and Spiegl-Kreinecker et al., 2015).The evidence for purifying selection in TERT-CLPTM1L region points to the importance of maintaining the structural integrity of this locus but also suggests that mechanisms other than protein altering mutations may play significant role such as interactions with other genes such as MYC (Koh et al., 2015) or miR-34a (Xu et al., 2015). The rationale for setting the threshold for marker surrogacy at r2 = 0.6 (p7) while using r2 = 0.8 for LD calculations (p3) should be explained. In summary, this is well designed and presented study that demonstrates the potential of using high throughput sequencing data together with growing resources such as ENCODE to enhance understanding of traditional genome-wide genotyping experiments. The title reflects well the contents, the abstract is appropriate and occlusions are justified and balanced.", "responses": [] } ]	1	https://f1000research.com/articles/3-231
https://f1000research.com/articles/3-196/v1	19 Aug 14	{ "type": "Research Article", "title": "Electrical maturation of neurons derived from human embryonic stem cells", "authors": [ "Michael Telias", "Menahem Segal", "Dalit Ben-Yosef", "Michael Telias", "Menahem Segal" ], "abstract": "In-vitro neuronal differentiation of human pluripotent stem cells has become a widely used tool in disease modeling and prospective regenerative medicine. Most studies evaluate neurons molecularly and only a handful of them use electrophysiological tools to directly indicate that these are genuine neurons. Therefore, the specific timing of development of intrinsic electrophysiological properties and synaptic capabilities remains poorly understood. Here we describe a systematic analysis of developing neurons derived in-vitro from human embryonic stem cells (hESCs). We show that hESCs differentiated in-vitro into early embryonic neurons, displaying basically mature morphological and electrical features as early as day 37. This early onset of action potential discharges suggests that first stages of neurogenesis in humans are already associated with electrical maturation. Spike frequency, amplitude, duration, threshold and after hyperpolarization were found to be the most predictive parameters for electrical maturity. Furthermore, we were able to detect spontaneous synaptic activity already at these early time-points, demonstrating that neuronal connectivity can develop concomitantly with the gradual process of electrical maturation. These results highlight the functional properties of hESCs in the process of their development into neurons. Moreover, our results provide practical tools for the direct measurement of functional maturity, which can be reproduced and implemented for stem cell research of neurogenesis in general, and neurodevelopmental disorders in particular.", "keywords": [ "human embryonic stem cells", "neural differentiation", "electrophysiology", "neuronal maturation", "synaptic activity", "action potential." ], "content": "Introduction\n\nIn-vitro neural differentiation (IVND) of human pluripotent stem cells (hPSCs), is a promising vehicle for disease modeling and regenerative medicine1–3. Several protocols for IVND of hPSCs including embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs), are used worldwide, resulting in different neuronal types4,5. Upon IVND, confirmation of the neuronal fate is commonly analyzed by the expression of neuron-specific genes, including those of cytoskeletal components (e.g.; TUJ1, MAP2), transcription factors (e.g.; NeuN, NeuroD1) and synaptic proteins (e.g.; synaptotagmin, synaptophysin)6. The expression of these genes indicates that the cell has acquired the machineries needed to build a neuron, but the ultimate indication that these are genuine neurons involves the analysis of their electrical properties. Most studies that do include electrophysiological recordings of hPSCs-derived neurons focus at one specific final time-point along the differentiation cascade, to demonstrate the neuronal identity that is related to the disease in study7–9. However, the analysis of the dynamics of electrical maturation at several time points can provide valuable insights into the pathology of neurodevelopmental disorders. Moreover, in the context of human embryonic neurogenesis, analyzing electrical maturation on hPSCs during IVND can shed light over molecular and cellular mechanisms that so far have been studied only using animal models.\n\nCurrently, only a few studies on IVND of hPSCs employed electrophysiological recordings at consecutive time points during neural differentiation, so far with inconsistent results, in terms of timing, frequency of action potentials and formation of spontaneous synaptic activity10–13. Although the timing of the development of neuronal electrical properties following IVND is extremely important, it currently remains poorly understood due to high variability in differentiation protocols, culture conditions and hPSC lines used. For this reason, the aim of this study was to systematically analyze, by electrophysiological tools, the developing neurons derived in-vitro from hESCs, in order to define predictive parameters for their electrical maturity, as well as to model the dynamics of neural development.\n\n\nMaterials and methods\n\nhESCs culture conditions and IVND: The hESC line HUES-13 (kindly provided by the Melton Lab, Harvard University), was used in all experiments. hESCs were cultured on feeder layers of mitomycin C (Sigma)-inactivated mouse embryonic fibroblasts in hES-medium supplemented with bFGF (R&D), as previously described7. Before induction of IVND, hESCs were cultured on Matrigel (BD)-coated wells for two passages. The dual SMAD inhibition IVND protocol was applied as previously described14, including minor modifications. Figure 1A illustrates the actual IVND protocol used. Briefly, neural induction was achieved by gradually changing the medium from hES to N2 while adding dorsomorphin and SB431542 for 10 days; neuronal induction was achieved by changing the medium to N2/B27 and adding BDNF, GDNF, ascorbic acid, dbcAMP and DAPT for 10 additional days. At day 20 cells were dissociated using Accutase (Life Tech.) and seeded on 13 mm glass coverslips previously coated with 50 µg/ml Poly-D-Lysine and 20 µg/ml Laminin (Sigma). Seeding density was ~1.0×105 cells/cm2. From day 20 and on, neurons were continuously grown in N2/B27 medium supplemented with 20 ng/ml BDNF, GDNF and NT3. Concentrations of reagents and growth factors used were as follows: 5 µM dorsomorphin (Stemgent), 10 µM SB431542 (Stemgent), 20 ng/ml BDNF (PeproTech), 20 ng/ml GDNF (PeproTech), 0.2 mM ascorbic acid (Sigma), 0.5 mM dbcAMP (Sigma), 10 µM DAPT (Tocris), 20 ng/ml NT3 (PeproTech). N2 medium was composed of DMEM:F12 (Life Tech.), supplemented with 1% N2 (Life Tech.), 1% non-essential amino acids (BioInd.), 1% Glutamax (Life Tech.) and 100 µg/ml Primocin (InvivoGen). N2/B27 Medium was a 1:1 mixture of N2 and B27 media. B27 medium was composed of Neurobasal (Life Tech.), supplemented with 1% B27 (Life Tech.), 1% non-essential amino acids (BioInd.), 1% Glutamax (Life Tech.) and 100 µg/ml Primocin (InvivoGen).\n\n(A) Schematic representation of the protocol for in-vitro neural differentiation using dual SMAD inhibition. The protocol includes 3 steps: 1) Neural induction (days 0–10), by blocking SMAD signaling using Dorsomorphin (DM) and the TGF-β inhibitor SB431542 while reducing the relative amounts of hES medium and concomitantly increasing the relative amounts of N2 medium. 2) Neuronal induction (days 10–20), by incubating cells with brain-derived neurotrophic factor (BDNF), glia-derived neurotrophic factor (GDNF), ascorbic acid, dibutyryl-cyclic-AMP (dbcAMP) and the NOTCH1-inhibitor DAPT; 3) Neuronal differentiation (days 20–37), by dissociating cells and re-plating them on poly-lysine/laminin-coated glass coverslips in the presence of BDNF, GDNF and neurotrophin 3 (NT3). The specific time-points for electrophysiological recordings are indicated with arrows. (B) Representative images of HUES-13 hESC line undifferentiated colonies stained positive for OCT4 (green); hESCs-derived neurons at day 21 stained positive for MAP2 (red) and hESCs-derived neurons at day 37 stained positive for NeuN (green). (C) Bright field representative images of neurons at days 23, 30 and 37 of in-vitro neural differentiation.\n\nCell patch-clamp: Electrophysiological recordings were conducted as previously described7. In brief, neurons on glass coverslips were transferred to a custom-made recording chamber adapted for an inverted microscope (Olympus XI-50), in standard recording medium, containing (in mM):10 HEPES, 4 KCl, 2 CaCl2, 1 MgCl2, 139 NaCl, 10 D-glucose (340 mOsm, pH 7.4). Cells were patch-clamped with glass pipettes (Sutter Instruments, 1.5 mm OD, 0.75 mm ID), pulled using a P87 Puller (Sutter Instruments). Pipettes contained intracellular medium composed of (in mM) 136 K-gluconate,10 KCl, 5 NaCl,10 HEPES, 0.1 EGTA, 0.3 Na-GTP, 1 Mg-ATP, and 5 phosphocreatine, pH 7.2 (pipette tip resistance was 5–8 MΩ). Action potentials were evoked (in current clamp mode) by injecting depolarizing current pulses. Membrane potential was held at -60 mV. Spontaneous synaptic currents were recorded in 2 minute sessions in voltage clamp mode with a 50 µs sampling rate. Signals were amplified with a Multiclamp700B amplifier and recorded with Clampex9.2 software (Axon Instruments). Data were subjected to a 500-Hz low-pass filter and analyzed using Clampfit-9 and SigmaPlot.\n\nImmunofluorescence: Immunostaining was performed as previously described7. Briefly, cells were fixated for 15 minutes at R.T. using Cytofix (BD) and washed with PBS. Primary antibodies were applied at 4°C, overnight, in a PBS solution containing 2.5% BSA and 0.1% Triton. Staining with secondary antibody was performed for 1 hour at R.T., in the dark. The pluripotent gene Oct4 was detected with monoclonal mouse anti-human OCT4 (Santa Cruz, #sc-5279, RRID: AB_628051, Lot C1308, dilution – 1:200). Neurons were stained using polyclonal rabbit anti-human MAP2 (Santa Cruz, #sc-20172, RRID: AB_2250101, Lot D2710, dilution 1:250), and monoclonal mouse anti-human NeuN (GeneTex, #GTX30773, RRID: AB_1949456, Lot 27334, dilution 1:20). Primary antibodies were detected using sheep anti-mouse Cy2-conjugated and goat anti-rabbit Cy3-conjugated secondary antibodies (Jackson Labs).\n\nImaging: Bright field and fluorescence images of cells were obtained using an Olympus IX51 inverted light microscope, and a Zeiss LSM 700 confocal microscope. Images were processed using Olympus CellA for XP (2006) and ImageJ (NIH, v. 1.49) software.\n\nStatistical analysis: Data were collected from 15–25 cells for each time point, in 2 different experiments. ANOVA was performed on data using SPSS (v. 19).\n\n\nResults\n\nWe have used a slightly modified version of an established protocol for IVND of hESCs5,14, that is based on the dual inhibition of the SMAD pathway (Figure 1A). SMADs are the mammalian homologues to drosophila mad and C. elegans sma, and function as cytoplasmic mediators of TGFβ signaling15. The process of IVND implemented here includes three major steps: (i) Neural induction by blocking SMAD signaling using Dorsomorphin and SB43154216,17; (ii) Neuronal induction by incubating cells with pro-neuronal factors (BDNF, GDNF, ascorbic acid, dbcAMP) and the NOTCH1-inhibitor DAPT18; (iii) Neuronal differentiation, by dissociating cells and re-plating them on poly-lysine/laminin-coated glass coverslips in the presence of BDNF, GDNF and NT3. Our hESCs selected for IVND were pluripotent as shown by the expression of OCT4 and the neurons derived were stained positive for MAP2 (>90% of cells), already at day 21 of IVND (Figure 1B). At day 37, >90% of cells expressed the neuronal transcription factor NeuN (Figure 1B). Typical neuronal morphology with 10–15 µm phase bright somata displaying pyramidal shape, extending an ‘apical’ dendrite and a few ‘basal’ dendrites was visible already at day 21 (Figure1B- MAP2, Figure 1C). Importantly, the neurons did not undergo any significant morphological changes during later stages of differentiation (Figure 1C, days 23, 30 and 37). Taken together, these results suggest that the cells analyzed during the three recording time-points (Figure 1A) are probably early human embryonic neurons, which are practically impossible to study in-vivo.\n\nCurrent clamp recordings of neurons at days 23, 30 and 37 of IVND (corresponding to days 3, 10 and 17 days following induction of neuronal differentiation), showed a steady increase in the excitability of hESCs-derived neurons (Figure 2A), while their input resistance remained similar for every time-point (Figure 2B-input resistance). At days 23 and 30, these neurons could discharge only single action potentials (APs; 21 and 25 neurons were recorded at days 23 and 30 respectively). However, multiple spikes were observed in all neurons recorded at day 37 (Figure 2A, B-spike frequency; 23 neurons were recorded). In addition, spike amplitude was significantly increased at day 30 and 37 as compared to day 23. Spike duration (measured at half-width of the action potential (AP), became shorter with prolonged differentiation, from 3.31±0.16 msec at day 23, to 2.57±0.10 msec at day 30, to a mean duration of 1.95±0.20 msec at 37 days (Figure 2B). On the other hand, spike threshold was significantly lower for day 37 only. After hyperpolarization (AHP) potentials were not detected at day 23, but were present at day 30 and 37, in which the drop from spike threshold was ~-7.9 mV for both time-points. These results clearly show a steady ongoing process of electrical maturation for human in-vitro developing neurons, in which sequential firing of multiple spikes is achieved by day 37 following induction of IVND with the dual SMAD inhibition protocol. Furthermore, our results show that spike frequency, amplitude, duration, threshold and after hyperpolarization can serve as the best predictive measurements for electrical maturation.\n\n(A) Representative traces for current clamp recordings at day 23 (black), 30 (red) and 37 (green). Membrane potential was held at ~-60 mV and voltage deflections (mV) are shown following 6 consecutive pulses of ~20 pA current injection from -40 to +60 pA. (B) Data analysis showing (from left to right): input resistance (GΩ), spike frequency (spike number per pulse), spike amplitude (mV), spike duration (msec), spike threshold (mV) and spike after hyperpolarization (‘AHP’, mV), for the same recordings days as in (A). Sample size: n=21 at day 23; n=25 at day 30; n=23 at day 37. Values are mean ± SEM. P<0.05, ANOVA.\n\nIn order to further explore the dynamics of time-dependent development of spike discharges, we examined properties of the derived neurons in voltage clamp mode at the same time-points listed above (Figure 3A). We measured K+ currents evoked by successive 20 mV voltage commands. The results show a significant difference between day 23 and 30 as compared to day 37 in the I-V curves of both the transient K+ (IA) current and the sustained (IK) current (Figure 3B; number of neurons recorded in each day was 21, 25 and 23 respectively). The sigmoid regression function that fits these curves is given by the equation: = a1+ex–xob, where ‘a’ is the slope of the curve. Our calculations show that the slope of the curve in IK increased from 1.36 (day 23), to 1.64 (day 30), and finally to 2.34 (day 37). Moreover, the value of a in IA increased from 1.49 (day 23), to 2.01 (day 30), and finally to 3.50 (day 37). This robust increase in the slope of both K+ currents from day 30 to day 37 is indicative that during this period the membrane of hESCs-derived neurons probably undergoes important changes in their expression of K+ channels. In addition to the parameters we have measured in current-clamp for APs characteristics, the slope of I-V curves of K+ currents could also be used as a tool to measure electrical maturity during IVND of hPSCs.\n\n(A) Representative traces for voltage clamp recordings at day 23 (black), 30 (red) and 37 (green). Membrane potential was held at -60 mV and current (nA) is shown following 12 consecutive pulses of 20 mV voltage steps from -100 to +60 mV. (B) I-V curves for IA and IK currents for the same time-points as in (A). Sample size: n=21 at day 23; n=25 at day 30; n=23 at day 37. Values are mean ± SEM.\n\nFinally, we examined the time-course of formation of spontaneous synaptic currents, indicating active synaptic connections (Figure 4A). No synaptic activity was found in any tested cell at day 23 (n=19). However, ~50% of neurons at day 30 and 37 showed spontaneous synaptic activity, which increased from day 30 to day 37 in both frequency and current amplitudes (number of neurons recorded was 18 and 23 respectively) (Figure 4B). No attempt was made to distinguish between excitatory and inhibitory synaptic currents in the present study. At day 37, spontaneous synaptic currents had a mean rise time of 1.84±0.16 msec, and their mean decay time was 3.25±0.71 msec. These results suggest that the capability of in-vitro hESCs-derived neurons to develop synaptic connections can arise at a relatively early time-point during IVND, and is concomitant with the developmental timing of burst firing. Furthermore, these results indicate that electrical maturation involves the development of intrinsic properties in individual neurons, in parallel with the development of network-activity.\n\n(A) Representative traces for spontaneous synaptic currents recorded in voltage clamp mode at -60 mV for days 23, 30 and 37. (B) Analysis of spontaneous synaptic currents frequency (Hz) and amplitude (pA). Sample size: n=19 at day 23; n=18 at day 30; n=23 at day 37. Values are mean ± SEM. P<0.05, ANOVA.\n\n\nDiscussion\n\nCurrently, there is no standard protocol to analyze the developmental stage in which all the required electrophysiological properties for proper function are already present in neurons derived by IVND of hPSCs. Several studies provide electrophysiological data on hPSCs-derived neurons but they do so only for a single specific end-point of the process, in order to compare control neurons to diseased ones7–9,19–26. However, the dynamic of electrical maturation at several time-points along the process has not been extensively investigated, and currently there are only a few studies which addressed this question, with inconsistent results10–13,27–29. Here, we have analyzed systematically the electrical maturation of human neurons derived from hESCs. We show that, by applying the dual SMAD inhibition in the IVND protocol on hESCs, early embryonic neurons are generated demonstrating electrical maturation already by day 37, including firing of spike bursts with increased amplitude and reduced duration. Our results further show that this electrical maturation is accompanied by a steady increase in K+ currents, which enabled faster and more reliable repolarization. Moreover, a steady and gradual increase in spontaneous synaptic activity is observed at the same three time-points, suggesting that electrical maturation occurs not in individual neuron, but also in the developing neuronal networks. Nevertheless, no spontaneous action potential discharges could be detected, indicating that the network is still not fully functional.\n\nAs shown here, the neurons we derived could fire trains of APs not before day 37 (~5 weeks) of IVND. Indeed, a study in which the same dual SMAD inhibition protocol was used to generate neurons, spike bursts were measured already by week 4, but this was observed only in ~40% of the hESCs-derived neurons12, as compared to our results demonstrating spike bursts in 100% of the neurons at the same time. In comparison, other studies have shown that when dopaminergic neurons or GABAergic interneurons were derived from hPSCs, a similar phenomenon of time-dependent electrical maturation was observed, but only following >8 weeks of IVND11,13,28. Furthermore, other IVND protocols applied on hESCs, showed no incidence of burst firing and no significant differences between time-points in APs parameters10,27. Our results indicate that measurement of spike frequency, amplitude, duration, threshold and after hyperpolarization can serve as predictive parameters for electrical maturity. Interestingly, spike duration was found to be the most reliable predictor parameter, and its measurement at each time-point tested. Similar to APs, K+ currents reflect the process of electrical maturation in a time-dependent manner. In addition, we have established that the slope of IK and IA steadily increases with time. Indeed, in the study of Takazawa et al., 2012, hESCs-derived spinal motor neurons demonstrating bursts of multiple APs at day 36, also showed a time-dependent maturation in the transient and in the sustained K+ currents (IA and IK, respectively,29). Furthermore, when neural differentiation through dual SMAD inhibition was performed to produce forebrain neurons, as we have done in this study, a time-dependent increase in the amplitude of both IA and IK was indeed shown during the first 4 weeks12. In contrast, Nicholas et al., 2013 showed in hPSCs-derived GABAergic interneurons a steady increase of an unspecified K+ current, reaching a maximal peak of ~1.5 nA only after 30 weeks of IVND13, but Hartfield et al., 2014 showed no significant changes between relevant time-points in the average peak amplitude of K+ currents in hPSCs-derived dopaminergic neurons27. These observations indicate that development of K+ currents takes place during the earliest stages of electrical maturation. However, more research is needed to understand how and when these K+ channels are expressed on the membranes of in-vitro developing neurons, and whether the increase in their current is caused by an increase in their density throughout the membrane or by maturation of their intrinsic activation properties.\n\nWe have shown here time-dependent development of spontaneous synaptic activity, at a relatively early time-point during IVND, and concomitant with the developmental timing of burst firing, indicating that electrical maturation involves the development of intrinsic properties in individual neurons, in parallel with the development of network-activity. Other studies however, have shown that although action potential can be produced at earlier stages of differentiation, the generation of neuronal networks as evidenced by spontaneous synaptic activity, is observed only at later stages of IVND, (at least 8 weeks of differentiation into GABAergic forebrain interneurons13,28, or 10 weeks of differentiation into dopaminergic midbrain neurons11). It has been suggested that IVND of hESCs produces immature embryonic-like neuronal cells, which take several months to develop the characteristic genetic and electrophysiological properties of mature adult-like neurons30,31. Others have proposed that IVND of hPSCs mimic the real time-frame of in-vivo human embryonic neurogenesis, due to an “intrinsic clock-like mechanisms”32. It is commonly accepted that synaptogenesis in humans starts only by the end of fetal life and during the first months of postnatal life6,33. Nevertheless, here we show significant spontaneous synaptic activity already by day 37, suggesting that in-vitro conditions results in an accelerated rate of maturation and development. The different timing of neuronal functional maturation observed in the different studies could be explained by high variability in cultures due to the different hPSCs used, different IVND protocols, different seeding density of cells, and other factors related to the IVND protocol used.\n\nIn conclusion, our results shed light on the dynamic development of the electrophysiological properties of individual neurons as well as in-vitro neuronal networks. Furthermore, these findings suggest critical electrophysiological parameters that can be used to predict the precise timing in which neuronal functionality is acquired by human cells developing in-vitro. Therefore, the results of the present study provide a valuable tool for the direct measurement of electrical maturity, which can be implemented when studying neurodevelopmental and neurodegenerative diseases.\n\n\nData availability\n\nfigshare: Data files electrical maturation of neurons derived from human embryonic stem cells doi: 10.6084/m9.figshare.113247534", "appendix": "Author contributions\n\n\n\nM.S. and D.B.Y. designed the study, wrote the manuscript and approved it. M.T. performed the experiments, collected and analyzed the data and wrote the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nM.T. was supported by the TEVA National Network of Excellency scholarship in neuroscience.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank Ms. Efrat Biton (Weizmann Inst.) for technical assistance and Dr. Michael Boland (Scripps Ins.) for valuable advice in differentiation procedures.\n\n\nReferences\n\nYu DX, Di Giorgio FP, Yao J, et al.: Modeling hippocampal neurogenesis using human pluripotent stem cells. Stem Cell Reports. 2014; 2(3): 295–310. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchwarz SC, Schwarz J: Translation of stem cell therapy for neurological diseases. Transl Res. 2010; 156(3): 155–60. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShi Y, Kirwan P, Smith J, et al.: A human stem cell model of early Alzheimer’s disease pathology in Down syndrome. Sci Transl Med. 2012; 4(124): 124ra29. PubMed Abstract \| Publisher Full Text\n\nChamberlain SJ, Lalande M: Neurodevelopmental disorders involving genomic imprinting at human chromosome 15q11-q13. Neurobiol Dis. 2010; 39(1): 13–20. PubMed Abstract \| Publisher Full Text\n\nChamberlain SJ, Chen PF, Ng KY, et al.: Induced pluripotent stem cell models of the genomic imprinting disorders Angelman and Prader-Willi syndromes. Proc Natl Acad Sci U S A. 2010; 107(41): 17668–73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBelinsky GS, Moore AR, Short SM, et al.: Physiological properties of neurons derived from human embryonic stem cells using a dibutyryl cyclic AMP-based protocol. Stem Cells Dev. 2011; 20(10): 1733–46. PubMed Abstract \| Publisher Full Text\n\nMaroof AM, Keros S, Tyson JA, et al.: Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells. Cell Stem Cell. 2013; 12(5): 559–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTakazawa T, Croft GF, Amoroso MW, et al.: Maturation of spinal motor neurons derived from human embryonic stem cells. PLoS One. 2012; 7(7): e40154. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaha K, Jaenisch R: Technical challenges in using human induced pluripotent stem cells to model disease. Cell Stem Cell. 2009; 5(6): 584–95. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu GH, Ding Z, Izpisua Belmonte JC: iPSC technology to study human aging and aging-related disorders. Curr Opin Cell Biol. 2012; 24(6): 765–74. PubMed Abstract \| Publisher Full Text\n\nMiller JD, Ganat YM, Kishinevsky S, et al.: Human iPSC-based modeling of late-onset disease via progerin-induced aging. Cell Stem Cell. 2013; 13(6): 691–705. PubMed Abstract \| Publisher Full Text\n\nHrvoj-Mihic B, Bienvenu T, Stefanacci L, et al.: Evolution, development, and plasticity of the human brain: from molecules to bones. Front Hum Neurosci. 2013; 7: 707. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTelias M, Segal M, Dalit BY: Data files electrical maturation of neurons derived from human embryonic stem cells. figshare. 2014. Data Source" }	[ { "id": "5864", "date": "01 Sep 2014", "name": "Maija Castren", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTelias et al. report a systematic analysis of intrinsic electrical properties of developing neurons differentiated from pluripotent human embryonic stem cells during neuronal maturation days 23-37 in vitro. Neuronal differentiation was performed by dual SMAD inhibition and differentiated neurons showed mature neuronal morhology. Current clamp recordings showed changes in spike amplitude, frequency and duration and sequential firing of multiple spikes was found by day 37. At the same time a significant increase in the frequency and amplitude of spontaneus synaptic activity could be detected and an augmentation of K+ currents. The provides new information about the dynamic development of the electrophysiological properties of neurons differentiated from pluripotent cells.The manuscript is well written and discussed. The title is appropriate for the paper. The methods are sound. The following points need to be addressed:Page 3, the second paragraph; it is not clear how images are indicated (Figure 1B-MAP2, Figure 1C). The figure 4B; Is the difference of frequency between values at day 30 and 37 significant with ? The presentation gives an impression that the difference is more significant.", "responses": [ { "c_id": "1010", "date": "28 Sep 2014", "name": "Michael Telias", "role": "Author Response", "response": "The authors wish to thank the referee for her kind words and the time to review our work.Specific comments:\"Page 3, the second paragraph; it is not clear how images are indicated (Figure 1B-MAP2, Figure 1C).”We have revised the text in the Results section “Differentiation process and neuronal morphology” to correct this mistake, as explained by the referee, and make the text more comprehensible: “[…] (MAP2 and NeuN in Figure1B and Figure 1C).” \"The figure 4B; Is the difference of frequency between values at day 30 and 37 significant with ? The presentation gives an impression that the difference is more significant.\"Indeed the p value in the frequency graph is <0.01, as noted by the referee. However, we chose to symbolize every p value that is below 0.05 with only one asterisk, regardless of its specific value." } ] }, { "id": "5862", "date": "24 Sep 2014", "name": "Shauna Yuan", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting article examining the time course of the electrophysiological properties of neurons derived from human embryonic stem cells. The authors studied firing of action potentials, potassium currents and spontaneous synaptic activities. They found that all of these properties gradually become more mature over time. This study shows the feasibility of conducting such experiments to map out developmental stages of neurons in a culture dish. The title and abstract convey the message of the manuscript. It is well-written and contains comprehensive information on the study design, methods and analysis. The data is well presented. It is not clear from the article that the three properties that the authors looked at are fully developed at 37 days. Can they comment on the variability between cultures? In the conclusion, the authors could possibly expand on what they think about the timing of maturation. Would the days of maturation be very different due to the differentiation method; therefore, such time-course should be performed for each new differentiation protocol?", "responses": [ { "c_id": "1011", "date": "28 Sep 2014", "name": "Michael Telias", "role": "Author Response", "response": "The authors wish to thank the referee for her kind words and the time to review our work.Specific comments:“It is not clear from the article that the three properties that the authors looked at are fully developed at 37 days. Can they comment on the variability between cultures?” The points raised by the referee are indeed interesting. Variability among cultures (different experiments with the same hESC line) was relatively low, as revealed by the standard error of the mean in the parameters measured, such as spike amplitude, duration, etc. (see Fig. 2B).Regarding the capability of cells of firing consecutive trains of action potentials: in this study we have recorded neurons up to day 37 following IVND, and concluded that these 37 days old neurons are already functionally mature. A continuous study, that we are currently conducting at our lab, demonstrates that in >60 days old neurons the only parameter that change, as compared to 37-days old neurons, is spike frequency but all other parameters remain the same (i.e., spike amplitude, duration, threshold, AHP). “In the conclusion, the authors could possibly expand on what they think about the timing of maturation. Would the days of maturation be very different due to the differentiation method; therefore, such time-course should be performed for each new differentiation protocol?”According to the referee’s comment, we have added the following explanation to the conclusion (last paragraph of Discussion): “Timing of electrical maturation can greatly vary among different cell lines of hESCs and hiPSCs, as well as between different protocols for IVND. Therefore, analysis of the parameters proposed in this study, which are universal for neuronal electrical activity and easy to reproduce, could serve to calibrate and adjust the time-course of electrical maturation in every cell line and for every protocol”" } ] } ]	1	https://f1000research.com/articles/3-196
https://f1000research.com/articles/3-230/v1	30 Sep 14	{ "type": "Research Article", "title": "The proteasome activity reporter GFP-Cl1 is degraded by autophagy in the aging model Podospora anserina", "authors": [ "Matthias Wiemer", "Heinz D. Osiewacz", "Matthias Wiemer" ], "abstract": "The degradation of damaged proteins is an important vital function especially during aging and stress. The ubiquitin proteasome system is one of the major cellular machineries for protein degradation. Health and longevity are associated with high proteasome activity. To demonstrate such a role in aging of Podospora anserina, we first analyzed the transcript and protein abundance of selected proteasome components in wild-type cultures of different age. No significant differences were observed. Next, in order to increase the overall proteasome abundance we generated strains overexpressing the catalytic proteasome subunits PaPRE2 and PaPRE3. Although transcript levels were strongly increased, no substantial effect on the abundance of the corresponding proteins was observed. Finally, the analysis of the P. anserina strains expressing the sequence coding for the CL1 degron fused to the Gfp gene revealed no evidence for degradation of the GFP-CL1 fusion protein by the proteasome. Instead, our results demonstrate the degradation of the CL1-degron sequence via autophagy, indicating that basal autophagy appears to be a very effective protein quality control pathway in P. anserina.", "keywords": [ "The degradation of proteins", "in particular of those that are damaged or are present in excess", "is an important vital function of biological systems and is implicated in several cellular processes such as cell cycle control", "proliferation", "differentiation", "apoptosis and protein quality control1. Impairments in protein degradation lead to the formation of protein aggregates2", "3", "promote the aging process4", "5 and convey the development of neurodegenerative diseases like Alzheimer’s or Parkinson’s disease6. There are two major pathways involved in protein degradation: autophagy and degradation by the ubiquitin proteasome system (UPS)7. Autophagy is effective in nutrient recycling and protein degradation. During autophagy proteins or whole organelles are engulfed by a double membrane forming autophagosomes that deliver their cargo to the lysosome in animals and the vacuole in plants and fungi8. The UPS consists of a large number of different ubiquitin ligases that act jointly with the proteasome", "a multi-protein complex with proteolytic activities. The ubiquitin ligases identify and mark proteins that need to be removed", "by formation of a chain of ubiquitin on the target protein9. A ubiquitin chain linked at K-48 is recognized by the 26S proteasome10. The 26S proteasome consists of two subcomplexes", "the catalytic 20S core particle and the 19S regulatory particle. The 19S regulatory particle conveys the identification", "deubiquitination", "unfolding and transport of the substrate into the proteolytic chamber. The core particle is responsible for the degradation of the target proteins. It is composed of four stacked rings", "which enclose the proteolytic chamber. The inner rings consist of 7 β-subunits", "including the proteolytic active PRE3 (β1)", "PUP1 (β2) and PRE2 (β5). The three catalytic subunits are the first substrates of the proteasome. Each contains a prosequence that is removed during assembly of the proteasome by an autocatalytic mechanism11", "12. The assembled β-subunits are framed by rings of seven α-subunits", "blocking the entrance to the proteolytic chamber", "if no regulatory particle is bound (reviewed in:13)." ], "content": "Introduction\n\nThe degradation of proteins, in particular of those that are damaged or are present in excess, is an important vital function of biological systems and is implicated in several cellular processes such as cell cycle control, proliferation, differentiation, apoptosis and protein quality control1. Impairments in protein degradation lead to the formation of protein aggregates2,3, promote the aging process4,5 and convey the development of neurodegenerative diseases like Alzheimer’s or Parkinson’s disease6. There are two major pathways involved in protein degradation: autophagy and degradation by the ubiquitin proteasome system (UPS)7. Autophagy is effective in nutrient recycling and protein degradation. During autophagy proteins or whole organelles are engulfed by a double membrane forming autophagosomes that deliver their cargo to the lysosome in animals and the vacuole in plants and fungi8. The UPS consists of a large number of different ubiquitin ligases that act jointly with the proteasome, a multi-protein complex with proteolytic activities. The ubiquitin ligases identify and mark proteins that need to be removed, by formation of a chain of ubiquitin on the target protein9. A ubiquitin chain linked at K-48 is recognized by the 26S proteasome10. The 26S proteasome consists of two subcomplexes, the catalytic 20S core particle and the 19S regulatory particle. The 19S regulatory particle conveys the identification, deubiquitination, unfolding and transport of the substrate into the proteolytic chamber. The core particle is responsible for the degradation of the target proteins. It is composed of four stacked rings, which enclose the proteolytic chamber. The inner rings consist of 7 β-subunits, including the proteolytic active PRE3 (β1), PUP1 (β2) and PRE2 (β5). The three catalytic subunits are the first substrates of the proteasome. Each contains a prosequence that is removed during assembly of the proteasome by an autocatalytic mechanism11,12. The assembled β-subunits are framed by rings of seven α-subunits, blocking the entrance to the proteolytic chamber, if no regulatory particle is bound (reviewed in:13).\n\nPrevious studies revealed that aging reduces the expression of genes coding for proteasome subunits and the activity of the proteasome in several model systems14–16. Also, several studies indicate a health and lifespan prolonging effects of high proteasome activity. For example, the proteasome activity is elevated in human fibroblast cell cultures derived from centenarians14 and in the liver of the naked mole rat17,18, a long-living rodent. Moreover, the overexpression of genes coding for proteasome subunit β1 or β5 in human fibroblasts was reported to lead to an increase in overall proteasome abundance and activity, resulting in an increased capacity to cope with stress19. Another component influencing proteasome activity is the proteasome assembly protein UMP1. Saccharomyces cerevisiae overexpressing ScUmp1 shows increased lifespan and viability in response to oxidative stress20. In S. cerevisiae, high levels of proteasome subunit ScRPN4 were reported to increase UPS capacity, enhance resistance to proteotoxic stress and increase replicative lifespan21. Overall, it appears that the proteasome is a relevant target for aging research. The data suggest that keeping protease activity high during aging can lead to an increase in the healthy lifespan of biological systems.\n\nWe use the filamentous ascomycete Podospora anserina as a model organism to investigate the mechanisms of aging including the role of different quality control pathways (for recent reviews see:22–24). In this study we investigated the impact of protein degradation by the UPS and autophagy. Although we could not demonstrate a role of the UPS, we established that the degradation of GFP-CL1 protein, that was expected to be a target of the proteasome, occurred via autophagy.\n\n\nMaterials and methods\n\nP. anserina was grown on plates with M2 medium (0.25 g/l KH2PO4 (Merck Cat# 5099.1000), 0.3 g/l K2HPO4 (Roth Cat# P749.1), 0.25 g/l MgSO4 × 7 H2O (Merck Cat# 1.05886.0500), 0.5 g/l urea (Merck Cat# 1.08487.0500) and 10 g/l yellow dextrin (Roth Cat# 6777.1), supplemented with 2.5 mg/l biotin (Serva Cat# 15060), 50 mg/l thiamine (Serva Cat# 36020), 5 mg/l citric acid × 1 H2O (Sigma-Aldrich Cat# C-0759), 5 mg/l ZnSO4 × 7 H2O (Merck Cat# Z-0625), 1 mg/l Fe(NH4)2(SO4)2 × 6 H2O (Merck Cat# 1.03861.0250), 2.5 mg/l CuSO4 × 5 H2O (Merck Cat# 2790.1000), 25 mg/l MnSO4 × 1 H2O (Serva Cat# 28405), 50 mg/l Na2MoO4 × 2 H2O (Serva Cat# 30207) and 50 mg/l H3BO3 (Merck Cat# 100165.5000) after sterilization of the basal medium) or in shaking Erlenmeyer flasks with CM-Medium (70 mM NH4Cl (Merck Cat# 1.01145.5000), 7.3 mM KH2PO4, (Merck Cat#1.04873.100), 6.7 mM KCl (Merck Cat# 1.04936.1000), 2 mM MgSO4 (Merck Cat# 1.05886.0500), 1% glucose (Sigma Cat# G-5400), 0.2% tryptone (Otto Nordwald Cat# 211701), 0.2% yeast extract (DIFCO Cat# 0127-07), 5 mM FeCl2 × 7 H2O (Merck Cat# 13478-10-9), 3.5 mM ZnSO4, (Merck Cat# 108883), 6.2 mM MnCl2, (Merck Cat# 5934.0100), pH 6.5) under constant light at 27°C. For germination, spores were incubated for two days in the dark on standard cornmeal agar supplemented with 60 mM ammonium acetate (Merck Cat# 1116.1000)25. Pieces of the mycelium derived from germinated spores were transferred on M2 medium to obtain cultures of specific age.\n\nAfter germination of spores, pieces of the mycelium were directly used or grown at 27° and constant light on M2 medium for 13 – 16 days (middle-aged) or 21 – 24 days (senescent), depending on the lifespan of the specific individual, to obtain cultures of specific age. A piece of the growth front was subsequently spread on a fresh M2 plate covered with cellophane (BioRad Cat# 1650963) and grown for 3 days. RNA was extracted with RNA-Plant kit (Machery-Nagel Cat# 740.949.250) and cDNA synthesis was performed using iScript kit (BioRad Cat# 170-8891). After dilution of cDNA to a concentration of 10 ng/µl, 20 ng was used per qRT-PCR reaction (IQ SybrGreen SuperMix, BioRad cat# 170-8882). The primers summarized in Table 1 were used to perform the qRT-PCR with three technical replicates per sample. A specific culture was compared to the mean CP of the juvenile cultures. Relative expression was normalized to PaPorin with the following formula26.\n\n\n\nE = PCR-Efficiency; CP = crossing point\n\nTo obtain total protein extracts, fungi of specific age were spread on a cellophane foil covered M2 surface for 3 days. Proteins were extracted as described in24. Briefly, the mycelia were harvested, ground under liquid nitrogen, mixed with extraction buffer and centrifuged at 14,000 g at 4°C for 10 min. The supernatant was recovered and used for the experiments. The protein extracts were fractionated by 2-phase SDS-PAGE (14% separating gels) according to standard protocol27. Proteins were subsequently transferred to PVDF membranes (Millipore Cat# IPFL00010). Blocking, antibody incubation and washing was performed according to western blot analysis handbook (LIC-OR Bioscience, Bad Homburg, Germany). The following primary antibodies were used: anti-PaPRE2 (rabbit, 1:500 dilution, raised against the specific peptide [H]-WKTKLEKGEFSNVT-[OH]; Sigma), anti-PaPRE3 (1:2500 dilution, raised against the specific synthetic peptide [H]-LYLPDTDYKVRHEN-[OH]; Sigma), anti-PaPUP1 (rabbit, 1:5000 dilution, raised against the specific peptide Ac-CLKRNYIKPNERT-amid, NEP), anti-HSP60 (mouse, 1:4000 dilution, Biomol, Cat# SPA-807), Anti-GFP (mouse, 1:10000 dilution, Sigma-Aldrich Cat# G6795 RRID:AB_563117). Secondary antibodies conjugated with IRDye 680 (1:15000 dilution, goat anti-mouse 680RD: LI-COR Biosciences Cat# 926-68070 RRID:AB_10956588) or IRDye CW 800 (1:15000 dilution, goat anti-rabbit 800: LIC-OR Biosciences, Cat# 926-3221) were used. After western transfer, the polyacrylamid gels were stained 1 h with coomassie blue as additional loading control.\n\nThe vector pExMthph28 was used as backbone for the generation of PaPre2, PaPre3 and PaUmp1 overexpression plasmids. For the assembly of pPaPre2Ex1, pExMthph was cut with BamHI and XbaI. The PaPre2 gene and terminator were amplified with the primers Pre2ExpFor (AAGGATCCATGGACACCCTCGTTGCG; restriction sites are underlined) and Pre2ExpXbaRev (AAAGATCTTGGCCCTCCTTACTAGAC), cut with BamHI and XbaI and ligated with the backbone. For the generation of pPaPre3Ex1, the PaPre3 gene and terminator were amplified with the primers PaPre3FwdBam (TTGGATCCATGGAATTCGGTACATCGGG) and PaPre3RevPst (TTCTGCAGCCCACAACCAGAACCTTTCAC) cut with BamHI and PstI and ligated with the similarly restricted vector pExMthph. pPaUmp1Ex1 was generated by amplification of PaUmp1, including the terminator, with the primers PaUmp1FwdBamHI (TTGGATCCATGGTAAGTTGCAGCCAACC) and PaUmp1RevPst1 (TTCTGCAGGCTCCCGTGAGGGCAGGAC), restriction of the product with BamHI and PstI, and ligation into the similarly cut vector pExMthph. The generation of a Gfp-Cl1 overexpression plasmid was performed by 3 fragment ligation. The Gpd promotor from Aspergillus nidulans, the eGfp gene and the first part of the Cl1-sequence were amplified by PCR with the plasmid pSM5 (based on pSM229) as template and with primers eGfp-Pgpd-cl1for (CCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATT) containing a HindIII restriction site and eGfp-Pgpd-cl1rev (GTGGCTAGC GCTGCTGAACCAGTTCTTGCAGGC CTTGTACAGCTCGTCCAT), containing half of the Cl1 sequence (italic) optimized for codon usage of P. anserina and a Eco47III restriction site. The second half of the Cl1-sequence was amplified with the TrpC terminator in a similar manner with the primers Ttrpc-cl1for (CTTCAGCGAG CTCAGCCACTTCGTCATCCACCTCTA ATCCACTTAACGTTACTGA) containing an Eco53kI restriction sites (underlined) and Ttrpc-cl1rev (CCACCGCGGTGGCGGCCGCTCTAGAAAGAAGGATTACCTC) containing a XbaI restriction site. The PCR products were ligated into the purified backbone of pSM5, previously cut with HindIII and XbaI. The plasmids were used to transform P. anserina wild-type spheroplasts according to24. pSM5 was used to generate a strain expressing Gfp without degron sequence. Briefly, mycelium of wild type “s” was blended and the cell wall digested with an enzyme solution. After filtration and concentration of sphaeroblasts by centrifugation, the sphaeroblasts were mixed with 10 µg plasmid DNA. Subsequently, polyethylene glycole (Serva Cat# 33136) was added to the sphaeroblasts. Transformants were selected for hygromycin B (Calbiochem Cat# 400051) resistance and the number of integrations was verified by Southern blot analysis.\n\nA piece of 2 day old mycelium was grown on a glass slide with a piece of PASM-medium27 covered with a coverslip and incubated at 27°C for 2 days. Heat stressed samples were incubated at 27°C for 24 h followed by an incubation at 37°C for 24 h. The cover slip with hyphae on it was visualized using a fluorescence microscope (DM LB, Leica, Wetzlar, Germany) with the appropriate excitation and emission filter to detect the GFP signal and a digital camera system (DC500, Leica, Wetzlar, Germany).\n\n\nResults\n\nIn order to address the role of the UPS on aging of P. anserina, we investigated the expression of the genes coding for proteolytic subunits PaPre2 (β5) and PaPre3 (β1) and of the proteasome assembly factor PaUmp1. First, we determined the abundance of transcript by using total RNA of juvenile, middle-aged and senescent cultures (Figure 1A). No significant changes in mRNA levels were observed in cultures of different age although mRNA abundance of all three genes was slightly reduced in senescent cultures. Next we analyzed protein levels of proteasome subunits in cultures of different age grown in standard growth medium and in medium to which paraquat was added as an inducer of oxidative stress. An increased abundance of mitochondrial HSP60 verified an increase in oxidative stress in senescent and in paraquat treated cultures (Figure 1B). However, no changes in the abundance of subunits PaPRE2, PaPRE3 and PaPUP1 (β3) of the proteasome were observed in the corresponding P. anserina cultures. We thus were unable to demonstrate a role of the ubiquitin proteasome system in counteracting adverse effects on cellular proteins in aged cultures and in cultures challenged with exogenous oxidative stress.\n\n(A) The expression of PaPre2, PaPre3 and PaUmp1 transcripts in juvenile, middle-aged and senescent samples is depicted relative to the juvenile wild type as mean ± SEM (5 – 7 biological replicates). (B) Western blot analysis of 50 µg total protein extracts of 5 d, 13 d and 21 d old wild type cultures grown on medium with and without the addition of 5 µM paraquat. Used antibodies are indicated on the right. The polyacrylamid gel stained with coomassie after blotting is used as loading control.\n\nHigh activity of the proteasome has been linked to increased health and lifespan30–33. In human cell cultures, the overexpression of the subunits β5 and β1 was found to increase the overall abundance of the proteasome, as well as its activity and resistance to oxidative stress34. To investigate whether or not such an effect is also observed in P. anserina, strains overexpressing the homolog subunits PaPre2 (β5) and PaPre3 (β1) were generated. First, plasmids conveying PaPre2 and PaPre3 overexpression were constructed and transformed into P. anserina spheroplasts (Table 2). Subsequently, overexpression of the genes was verified by qRT-PCR. PaPre3 expression was increased by factor 140 to 380 in the respective overexpression strain compared to wild type (Figure 2A). The PaPre2 overexpression strain exhibited a 94 times higher PaPre2 expression than the wild type (Figure 2B). In the next step, we evaluated protein levels in the overexpression strains by western blot immunodetection. The analysis of three independent PaPre3 overexpressors revealed two strains with unchanged protein abundance and one strain (PaPre3_OEx2) in which increased PaPRE3 signals occurred (Figure 2C). However, the detected signals are larger or smaller than expected for processed PaPRE3 and probably represent unprocessed PaPRE3 and a degradation product. A strain overexpressing PaPre2 showed no increase in PaPRE2 abundance compared to the wild type (Figure 2D). Thus, despite the strong increase in mRNA abundance, no substantial change in protein levels of the two investigated proteasome subunits was observed in the generated strains.\n\nThe expression of PaPre3 (A) and PaPre2 (B) in the respective overexpression strain was examined by qRT-PCR. (C, D) Total protein extracts of PaPre3 (50 µg) and PaPre2 (60 µg) overexpression strains were probed with α-PaPRE3 and α-PaPRE2 for the amount of processed proteasome subunits. The polyacrylamid gel stained with coomassie after blotting is displayed as loading control.\n\nThe activity of the UPS is not exclusively defined by the abundance of proteasome subunits but influenced by various factors like ubiquitin ligases, deubiquitin ligases, ATP-level, the regulatory particle, oxidative stress and post-translational modifications. In order to evaluate the efficiency of the UPS during aging of P. anserina, we generated two Gfp-cl1 strains with similar properties and a Gfp strain. Successful transformation of wild type was verified by Southern blot analysis (Table 2). The introduced genes are under the control of the constitutive Gpd promoter of Aspergillus nidulans. Gfp-cl1 codes for a protein containing the CL1 degron sequence fused to GFP. The CL1 sequence is a part of the Saccharomyces cerevisiae genome. It was first described in a ScURA3-CL1 fusion protein, which is unstable in wild type, but stable in strains lacking the ubiquitin ligases ScUbc6 and ScUbc7. Due to these characteristics the Cl1 degron has been used to monitor proteasome activity in various species including fly35, mouse36, rat37,38 and human cell cultures39–44.\n\nIn our work, we investigated the degradation of the CL1 degron fused to GFP. Fluorescence microscopy revealed diffuse fluorescence in whole cells of strains expressing Gfp-cl1 and Gfp, respectively, indicating a cytoplasmic localization (Figure 3A). Significantly, after applying heat stress, the Gfp-cl1 strain revealed a vacuolar localization of the GFP signal in some parts of the mycelium (Figure 3A). Western-blot analysis revealed two distinct GFP signals in Gfp-cl1-strains (Figure 3B). One signal corresponds to a protein with a size of 28.8 kDa expected for GFP-CL1 fusion protein while the other has the size of free GFP (26.9 KDa). This result was surprising, because proteasomal degradation should result in total decomposition of GFP-CL1 and provided a first clue for the degradation of the CL1 degron sequence by autophagy since the GFP part remains and is not, or only slowly, degraded by vacuolar proteases17,18. To verify the degradation of the CL1 degron by autophagy, we generated a P. anserina strain lacking PaAtg145 and expressing Gfp-cl1 by crossing of single mutants and selection of the double mutant. PaATG1 is necessary for autophagy and ΔPaAtg1-strains are not able to transport proteins to the vacuole for degradation45. Western blot analysis revealed that the double mutant contains only GFP-CL1 and no free GFP (Figure 3C), demonstrating that GFP-CL1 is at least partially degraded via autophagy in the wild type of P. anserina. This conclusion is supported by the accumulation of green fluorescence in the vacuoles of Gfp-cl1 overexpressing strains after the induction of autophagy by heat stress (Figure 3A).\n\n(A) Fluorescence microscopy analysis of Gfp and Gfp-cl1-1 mutant. Fungi were grown for 2d at 27°. Heat stressed samples were incubated at 37°C for the last 24 h. (B) Western-blot analysis of 60 µg total protein extracts from Gfp-Cl1-1 and Gfp strains. Proteins were detected by western-blot-analysis with α-GFP antibody. The corresponding polyacrylamid gel was stained with coomassie after blotting as loading control. (C) Western-blot analysis of 45 µg total protein extracts from Gfp-cl1-2 strains and from a Gfp-cl1-2/ΔPaAtg1 double mutant. Proteins were detected by western-blot-analysis with α-GFP antibody. The polyacrylamid gel was stained with coomassie after blotting.\n\n\nDiscussion\n\nIn the current study, we investigated the role of the proteasome in aging of P. anserina. Contrary to the mammalian model systems, we did not detect significant reduction of transcript or protein levels of proteasome subunits during aging46. Moreover, attempts to modulate the abundance of selected proteasomal subunits failed, although transcript abundance was strongly increased in the generated overexpression strains. It appears that in P. anserina the biosynthesis of the investigated proteasome subunits is under a strong post-transcriptional control.\n\nOne aim of our study was the development of an assay to study proteasomal activity. In other systems such assays are based on the microscopic monitoring of fluorescence changes resulting from the degradation of a reporter protein, termed degron, which is fused to GFP. The degron becomes rapidly ubiquitinated and subsequently the whole fusion protein is delivered to the proteasome were it is degraded. A widely used degron is CL1 derived from S. cerevisiae and consisting of 15 hydrophobic amino acids. Although this sequence was successfully used to detect proteasome activity in a wide range of organisms including yeast47, fly35, mouse36, rat37,38 and human cell cultures39–44, our experiments did not reveal a clear degradation of the whole GFP-CL1 fusion protein as it would be expected for degradation by the proteasome. Beside other reasons, it may be that CL1 is not recognized by the P. anserina ubiquitination system and thus does not constitute a functional degron. On the other hand, the sequence may be recognized by both the UPS and the autophagy machinery. Under the investigated conditions autophagy may be by far more efficient than ubiquitination and proteasomal degradation. An overlap of UPS and autophagy substrates has been shown previously48–51. The degradation of this reporter by autophagy may indeed be a severe problem for the establishment of a reporter gene based proteasome activity assay in filamentous fungi because they seem to be characterized by high level of basal autophagy. In Aspergillus oryzea, mitochondria, peroxisomes and nuclei of basal hyphae are degraded during normal growth in an autophagy dependent manner to use the nutrients to support growth52. Previous work in P. anserina also detected a high basal autophagy level under non-starved standard growth conditions45. On the other hand, basal autophagy in yeast and mammalian cell cultures appears to be low53,54. Another complication of the system may be that, as previously shown in Caenorhabditis elegans and neuronal rat cells, CL1 fused to GFP can form toxic aggregates if the expression level exceeds the capacity of the degradation system55. In our experiments this latter problem appears not to be valid since the fluorescence signal is distributed throughout the cell although we detected small condensed signals in some cells, which could indicate the formation of protein aggregates. Since such aggregates were only very small spots compared to those demonstrated in the mentioned studies with C. elegans and rat cells, the formation of toxic GFP-CL1 aggregates appears to be negligible under the chosen expression conditions.\n\n\nData availability\n\nfigshare: Raw data of qRT-PCR and western blot analyses of proteasome subunits and GFP-CL1 degradation in Podospora anserine. DOI: 10.6084/m9.figshare.117791056", "appendix": "Author contributions\n\n\n\nMW performed experiments. HDO initiated and supervised this study. HDO and MW wrote the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nIn part, this work was supported by a grant of the European Commission (Acronym: Proteomage, FP6-51830) to HDO.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank Dr. J. Servos (Frankfurt) for providing plasmids used in this study and Dr. A. Hamann for discussion of data.\n\n\nReferences\n\nNaujokat C, Hoffmann S: Role and function of the 26S proteasome in proliferation and apoptosis. Lab Invest. 2002; 82(8): 965–980. PubMed Abstract \| Publisher Full Text\n\nCummings CJ, Reinstein E, Sun Y, et al.: Mutation of the E6-AP ubiquitin ligase reduces nuclear inclusion frequency while accelerating polyglutamine-induced pathology in SCA1 mice. Neuron. 1999; 24(4): 879–892. 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Data Source" }	[ { "id": "6268", "date": "14 Oct 2014", "name": "Nektarios Tavernarakis", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript aims to address the role of the proteasomal activity in aging and health span, using Podospora anserina as a model organism. The authors show that gene expression and protein abundance of proteasome subunits and proteasome assembly factor remain stable during aging and oxidative stress. The authors used strains overexpressing the catalytic proteasome subunits PaPRE2 and PaPRE3 to increase proteasomal activity, but found no difference in lifespan, suggesting that proteasome activity does not affect lifespan. The study would benefit by including proteasomal activity controls to confirm activity increase. This is particularly relevant since the positive effect of the proteasome in lifespan, senescence and healthspan has been convincingly demonstrated in other systems. The authors suggest that the reporter they use, GFP-Cl1, is processed by the autophagic machinery. It would be interesting to examine whether overexpression of this construct causes aggregation of GFP which in turn triggers autophagy that subsequently clears the aggregates which cannot be cleared by the proteasome. As the authors state in their discussion toxic aggregates could be formed and this possibility should be experimentally evaluated. For instance the authors should evaluate whether ERAD (endoplasmic reticulum associated protein degradation) takes place, since accumulation of protein aggregates could initiate ER stress and ERAD. It is known that under various stress conditions proteasomal activity is enhanced. Perhaps the authors should try other conditions in addition to oxidative stress (ER stress, heat stress etc.).", "responses": [] }, { "id": "6542", "date": "03 Dec 2014", "name": "Suresh Rattan", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very well written paper dealing with the determination of the proteasome activity in the ageing model Podospora. The experimental work is technically sound and is performed diligently. Although the results obtained, especially with respect to the lack of change in the protesomal activity during ageing of Podospora, appear to be contrary to the ones reported for mammalian systems and for Drosophila, it does not decrease the significance of this study which once again underlies the fact that age-related changes can be both universal (public) or specific (private). Actually, this is the important point that needs to be discussed in this paper as to why such differences are observed (see some papers by George Martin on public and private mechanisms of ageing). Authors should therefore comment on the usefulness and limitations of using model systems such as Podosopora in the context of modulating human ageing and longevity, and for testing/screening potential ageing-modulatory strategies. Furthermore, it will be useful if the implications of enhanced autophagy-related pathways in the context of ageing are discussed a bit more.On a minor note, just to be up-to-date with the literature on proteasome activity during ageing of various systems (refs 14-16), authors may include and cite a paper on Drosophila (Hansen et al., 2012) which deals with the sex-specific differences in proteasome activity changes during ageing etc.", "responses": [ { "c_id": "1184", "date": "16 Jan 2015", "name": "Heinz Osiewacz", "role": "Author Response F1000Research Advisory Board Member", "response": "We appreciate very much your careful reading of the manuscript and the submitted comments. We totally agree that work on model systems has to be taken with caution. But this is inherent to ANY model including whole organisms (both lower as well as higher systems like mice) as well as in vitro systems like mammalian cell cultures. Nevertheless, models can provide important general information about the aging process in humans. This has been demonstrated in P. anserina which in the past has proven to generate pioneer knowledge on age-related processes like mitochondrial DNA instabilities 1,2, the role of mitochondrial dynamics 3, or mitochondrial ultrastructure 4.However, we would like to emphasize that the aim of our work reported in this F1000Research paper was to evaluate the role of the proteasome in P. anserina. Following different approaches, we could not demonstrate such a role. We are aware that we do not exclude a role of the proteasome (in fact, we believe that it also plays a role). The key information we wanted to pass on to the scientific community is that autophagy acts on a substrate, the CL1 degron, that in other systems has been shown to be degraded by the proteasome and that autophagy plays is highly active in the system. We strongly believe that the work we presented here exactly meets the aims and scope of F1000Research. References 1. Esser K, Tudzynski P, Stahl U, Kuck U: A Model to Explain Senescence in the Gilamentous Fungus Podospora anserina by the Action of Plasmid Like DNA. Molec gen Genet. 1980; 178 (1): 213-216 Reference Source 2. Stahl U, Lemke PA, Tudzynski P, Kuck U, et al.: Evidence for plasmid like DNA in a filamentous fungus, the ascomycete Podospora anserina. Molec Gen Genet. 1978; 162 (3): 341-343 PubMed Abstract3. Scheckhuber CQ, Erjavec N, Tinazli A, Hamann A, et al.: Reducing mitochondrial fission results in increased life span and fitness of two fungal ageing models. Nat Cell Biol. 2007; 9 (1): 99-105 PubMed Abstract \| Publisher Full Text \| Reference Source 4. Daum B, Walter A, Horst A, Osiewacz HD, et al.: Age-dependent dissociation of ATP synthase dimers and loss of inner-membrane cristae in mitochondria. Proc Natl Acad Sci USA. 2013; 110 (38): 15301-15306 PubMed Abstract \| Free Full Text \| Publisher Full Text \| Reference Source" } ] }, { "id": "6891", "date": "09 Dec 2014", "name": "Thorsten Hoppe", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is well established that proteasomal degradation declines during aging in different cell culture systems and model organisms. Therefore, it is of general interest whether such activity changes are conserved throughout evolution. Here, the authors analyzed mRNA levels of the 20S proteasomal core subunits PaPre2, PaPre3, and the proteasomal assembly factor PaUmp1 as well as protein levels of PaPRE2, PaPRE3, and PaPUP1 in the fungus P. anserina. The experiments are convincing and well described. Surprisingly, the authors did not find any age-dependent differences with respect to the investigated proteasomal factors. However, as measuring proteasomal activity directly did not work, it remained unclear whether a decline in protein turnover is directly linked to aging in this model organism. As highlighted by the authors, other factors including ubiquitin ligases, deubiquitylation enzymes, the 19S regulatory particle, or post-translational modifications of these factors might be even more limiting for ubiquitin-dependent degradation and regulated with age in the fungus. In this context the authors could have discussed the finding that in C. elegans and human stem cells overexpression of the regulatory subunit RPN-6 is sufficient to reduce the amount of toxic protein aggregates and increase life span (Vilchez et al. 2012).The authors state that protein levels of PaPUP1 did not change during aging and paraquat treatment. However, western blot analysis of figure 1B shows increased PaPUB1 levels in 21 day old cultures that were treated with paraquat compared to either paraquat treated young cultures or untreated old cultures. This does not necessarily reflect an age- or stress-dependent increase in PaPUB1 levels in P. anserina, but could be explained by a selective survival of fungi with higher PaPUB1 levels under toxic stress conditions.This study nicely confirms the impression that P. anserine exhibits high basal rates of autophagy in contrast to other organisms and thus might serve as an excellent model organism for studying the role of autophagy under different physiological conditions. Given the elevated level of autophagy, this organism however might not be optimal for the investigation of age-dependent changes in the ubiquitin/proteasome system that is relevant for higher organisms.", "responses": [] }, { "id": "6929", "date": "29 Dec 2014", "name": "Tilman Grune", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting investigation studying proteostasis in Podospora during aging. I am not a specialist in Podospora, but can judge the work on aging and the proteolytic systems.I believe that this paper is lacking one substantial part: this is the measurement of activities of the proteolytic systems. Without this it is hard to draw any conclusion. Major points:In the introduction the authors refer to references reporting “reduced expression of genes for proteasome subunits and the activity of the proteasome” in other aging models. This is true, but the authors should note that the only consistent finding in the literature is a reduced proteasomal activity. Other authors also reported minimal or no changes in proteasomal gene expression, but a reduced activity of the proteasome. Therefore, the measurement of the proteasomal activity is of utmost importance. The conclusion (page 4) that the proteasome has no “role…counteracting adverse effects on cellar proteins” is not supported by the data at all, without activity measurement, inhibition of the proteasome and so on. In Fig.2 the authors refer to “..processed protein” What is this? The number of translation products produced? How can this be concluded, since the authors just study the steady state level? Another statement on page 5: “..proteasomal degradation should result in a total decomposition of GFp-CL1”. In the light of the work of DeMartino/Thomas (Liu et al. 2003), I am not sure, whether this is true under all conditions. The knockout of ATG1 will reduce the macroautophagy process. (Are there alternative pathways in Podospora?) Is in the ATG1 k.o. the proteasome still active? Activities should be measured here, again. The formation of GFP-based aggregates is discussed by the authors. However, also here it is required to show that the proteasomal system is active, since low amounts of protein aggregates might be able to block/inhibit substantial parts of the proteasome.Minor points:In the introduction the authors refer to autophagy as parts of the cell “engulfed by a double membrane”. This is only true for macroautophagy, not for chaperone-mediated autophagy and microautophagy. I do not know, whether these other forms exist in Podospora, but since the authors in their argumentation are jumping in the whole paper from model to model, this should be corrected.", "responses": [ { "c_id": "1183", "date": "16 Jan 2015", "name": "Heinz Osiewacz", "role": "Author Response F1000Research Advisory Board Member", "response": "We thank you very much for careful reading of the manuscript and your comments and suggestions. We agree that it would be very helpful to include the biochemical in vitro assay to measure proteasome activity in protein extracts. Ideally, both in vitro and in vivo studies about the activity of the proteasome should be performed. In fact, the isolation of functional proteasomes in filamentous fungi with a rigid cell wall appears to be a problem. We therefore tried to use the in vivo degradation of the CL1-degron in our study. We would like to stress here that we do not state that protein degradation via the proteasome does NOT play a role in P. anserina. We just cannot demonstrate this with the available methods, and this is what we wanted to report. Apart from this, we identify that the GFP-CL1-degron is degraded to GFP in an ATG1-dependent manner. The resulting free GFP is resistant to degradation. The knock-out of an essentiell autophagy component, like ATG1, is the accepted method to demonstrate a role of autophagy. We show that GFP-fluorescence ends up in the vacuole. These are new findings that, together with our recent work 1, stress the importance of autophagy in the Podospora anserina system. References 1. Knuppertz L, Hamann A, Pampaloni F, Stelzer E, et al.: Identification of autophagy as a longevity-assurance mechanism in the aging model Podospora anserina.Autophagy. 2014; 10 (5): 822-834 PubMed Abstract \| Publisher Full Text \| Reference Source" } ] } ]	1	https://f1000research.com/articles/3-230
https://f1000research.com/articles/3-160/v1	11 Jul 14	{ "type": "Correspondence", "title": "Non-human lnc-DC orthologs encode Wdnm1-like protein", "authors": [ "Johannes M. Dijkstra", "Keith T. Ballingall", "Keith T. Ballingall" ], "abstract": "In a recent publication in Science, Wang et al. found a long noncoding RNA (lncRNA) expressed in human dendritic cells (DC), which they designated lnc-DC. Based on lentivirus-mediated RNA interference (RNAi) experiments in human and murine systems, they concluded that lnc-DC is important in differentiation of monocytes into DC. However, Wang et al. did not mention that their so-called “mouse lnc-DC ortholog” gene was already designated “Wdnm1-like” and is known to encode a small secreted protein. We found that incapacitation of the Wdnm1-like open reading frame (ORF) is very rare among mammals, with all investigated primates except for hominids having an intact ORF. The null-hypothesis by Wang et al. therefore should have been that the human lnc-DC transcript might only represent a non-functional relatively young evolutionary remnant of a protein coding locus. Whether this null-hypothesis can be rejected by the experimental data presented by Wang et al. depends in part on the possible off-target (immunogenic or otherwise) effects of their RNAi procedures, which were not exhaustive in regard to the number of analyzed RNAi sequences and control sequences. If, however, the conclusions by Wang et al. on their human model are correct, and they may be, current knowledge regarding the Wdnm1-like locus suggests an intriguing combination of different functions mediated by transcript and protein in the maturation of several cell types at some point in evolution. We feel that the article by Wang et al. tends to be misleading without the discussion presented here.", "keywords": [ "In their recent publication in Science", "Wang et al.1 aimed to identify lncRNAs involved in DC differentiation and function. In order to do this they used an established model of human DC differentiation from peripheral blood monocytes (Mo)", "based on addition of recombinant cytokines. They found that transcription of the human Wdnm1-like pseudogene (Wdnm1-like-ψ)", "or lnc-DC as they call it", "was robustly induced by the Mo-DC differentiation process. Furthermore", "they found Wdnm1-like-ψ highly transcribed in other dendritic cells", "and confirmed correlation of Wdnm1-like-ψ transcription with DC differentiation in several ways. To investigate a functional role of Wdnm1-like-ψ in their Mo-DC differentiation model", "they used a lentivirus-mediated RNA interference (RNAi) system. The RNAi interference with Wdnm1-like-ψ fragments resulted in a pronounced effect on Mo-DC differentiation as measured by expression of genes and molecules involved in the immune system", "the ability to take up antigen", "and the capacity to stimulate T-helper cells. Wang et al. showed by a number of experiments that the Wdnm1-like-ψ transcript", "in particular the 3’-end", "has some specificity for binding to the STAT3 transcription factor and can reduce STAT3 dephosphorylation by phosphatase SHP1. And", "importantly", "they showed that in their human Mo-DC differentiation model the effect of STAT3 inhibition caused similar effects as knockdown of Wdnm1-like-ψ. They therefore postulated that human Wdnm1-like-ψ transcript is an important regulator of DC differentiation by enhancing STAT3 activity through prevention of STAT3 dephosphorylation by SHP1. The results and human model presented by Wang et al. are generally convincing", "yet some questions remain", "such as to why not for all experiments both “no transfection control” (used in a few experiments) and “control RNAi” (used in all experiments) were included", "and why they only used a single RNAi control sequence. RNAi control sequences are relevant because off-target genes might be knocked down (e.g. Jackson et al.3)", "but also because the lentivirus system using short hairpin RNA (shRNA) can have immunogenic properties in an shRNA-sequence-dependent manner (e.g. Kenworthy et al.4). Notably", "in some experiments Wang et al.1 independently knocked down two different fragments of Wdnm1-like-ψ", "with similar experimental results", "thus reducing the chance that off-target effects of their RNAi systems influenced their conclusions. On the other hand", "since the use of two positive RNAi systems suggests that Wang et al. were aware of the potential weaknesses of the system", "this raises the question as to why they only used a single sequence for their RNAi control experiments. Regardless", "we consider the part of their manuscript on human Wdnm1-like-ψ to be mostly solid and interesting", "and the main reason why we are so (overly) critical is that acceptance of the model for human Wdnm1-like-ψ function as proposed by Wang et al. leads to a quite spectacular evolutionary model", "as outlined below." ], "content": "Correspondence\n\nIn their recent publication in Science, Wang et al.1 aimed to identify lncRNAs involved in DC differentiation and function. In order to do this they used an established model of human DC differentiation from peripheral blood monocytes (Mo), based on addition of recombinant cytokines. They found that transcription of the human Wdnm1-like pseudogene (Wdnm1-like-ψ), or lnc-DC as they call it, was robustly induced by the Mo-DC differentiation process. Furthermore, they found Wdnm1-like-ψ highly transcribed in other dendritic cells, and confirmed correlation of Wdnm1-like-ψ transcription with DC differentiation in several ways. To investigate a functional role of Wdnm1-like-ψ in their Mo-DC differentiation model, they used a lentivirus-mediated RNA interference (RNAi) system. The RNAi interference with Wdnm1-like-ψ fragments resulted in a pronounced effect on Mo-DC differentiation as measured by expression of genes and molecules involved in the immune system, the ability to take up antigen, and the capacity to stimulate T-helper cells. Wang et al. showed by a number of experiments that the Wdnm1-like-ψ transcript, in particular the 3’-end, has some specificity for binding to the STAT3 transcription factor and can reduce STAT3 dephosphorylation by phosphatase SHP1. And, importantly, they showed that in their human Mo-DC differentiation model the effect of STAT3 inhibition caused similar effects as knockdown of Wdnm1-like-ψ. They therefore postulated that human Wdnm1-like-ψ transcript is an important regulator of DC differentiation by enhancing STAT3 activity through prevention of STAT3 dephosphorylation by SHP1. The results and human model presented by Wang et al. are generally convincing, yet some questions remain, such as to why not for all experiments both “no transfection control” (used in a few experiments) and “control RNAi” (used in all experiments) were included, and why they only used a single RNAi control sequence. RNAi control sequences are relevant because off-target genes might be knocked down (e.g. Jackson et al.3), but also because the lentivirus system using short hairpin RNA (shRNA) can have immunogenic properties in an shRNA-sequence-dependent manner (e.g. Kenworthy et al.4). Notably, in some experiments Wang et al.1 independently knocked down two different fragments of Wdnm1-like-ψ, with similar experimental results, thus reducing the chance that off-target effects of their RNAi systems influenced their conclusions. On the other hand, since the use of two positive RNAi systems suggests that Wang et al. were aware of the potential weaknesses of the system, this raises the question as to why they only used a single sequence for their RNAi control experiments. Regardless, we consider the part of their manuscript on human Wdnm1-like-ψ to be mostly solid and interesting, and the main reason why we are so (overly) critical is that acceptance of the model for human Wdnm1-like-ψ function as proposed by Wang et al. leads to a quite spectacular evolutionary model, as outlined below.\n\nWhereas the presentation of their human data appears to be mostly correct, we feel that the way Wang et al.1 present their mouse model is inappropriate. Wang et al. used a mouse model to confirm that knockdown of Wdnm1-like(-ψ) results in impaired DC differentiation. Technically these experiments in mice worked as they expected, indicated both by in vitro and in vivo results, and they also found that knockdown of murine Wdnm1-like could lead to reduction of STAT3 phosphorylation, although they apparently did not check if murine Wdnm1-like transcript can bind STAT3. However, even though Wang et al. refer to Gene symbol 110000G20Rik which mentions “Wdnm1-like”, they only present the readers with the term “mouse lnc-DC ortholog”. This is highly misleading as it suggests that the transcript also relates to a long noncoding RNA in mice. The authors even state “Taken together, our data suggest that lnc-DC is vital for DC differentiation in both human and mice”. However, in mice the gene encodes a functional Wdnm1-like protein2, and our extensive analysis of mammalian sequence databases indicates that the Wdnm1-like ORF incapacitation is very rare among mammals. Actually, among the eutherian mammals that we investigated and for which the relevant genomic region information was available, only humans (and Neanderthals and Denisovans) lacked the capacity to encode the otherwise highly conserved Wdnm1-like protein sequence (Figure 1). At the level of the genus Pan (chimpanzee and bonobo) the N-terminus of the predicted mature protein differs from consensus, but even in gorilla and orangutan the encoded Wdnm1-like protein appears fully normal. So possibly the function of the Wdnm1-like protein started to lose importance after separation of Homo/Pan from the other apes, which is quite recent in evolutionary terms. Calculation of synonymous (ds) versus nonsynonymous (dn) nucleotide substitution rates, using software available at http://www.hiv.lanl.gov/content/sequence/SNAP/SNAP.html, indicates conservation of Wdnm1-like protein function after most of the animals shown in Figure 1 had separated in evolution. Namely, in pairwise comparisons, for the depicted set of eutherian mammals except Pan/Homo the average ds/dn ratio is 3.5, and for the set of primates except Pan/Homo this value is 3.0. Thus, although in each individual species experimental evidence would still be required, it is expected that most eutherian mammals possess functional Wdnm1-like protein.\n\nThe figure shows deduced Wdnm1-like amino acid sequences plus their coding nucleotide sequences in representative mammals.\n\nAfter evolutionary separation from gorilla, in an ancestor common to the genera Pan (including chimpanzee and bonobo) and Homo (including human, Neanderthal and Denisovan), the nucleotide region coding the N-terminus of the mature Wdnm1-like protein was modified by deletions (yellow shading). Nevertheless, in the genus Pan the Wdnm1-like open reading frame (ORF) remained intact. Only in Homo the Wdnm1-like coding sequence was interrupted by a frameshift through a single nucleotide deletion (red shading) within the leader peptide coding region (the resulting change in amino acids is shaded grey). For the human Wdnm1-like locus several transcripts (splicoforms) were found (Ensembl reports ENST00000590346, ENST00000588180, ENST00000587298, ENST00000590012, ENST00000589987, ENST00000592556, ENST00000566140, and ENST00000589777); however, we agree with Wang et al.1 that software investigation of the known transcripts suggests that the human Wdnm1-like locus does not code a functional protein (analyses not shown).\n\nThe marsupial Monodelphis domestica (opossum) was the only non-eutherian mammal for which we could identify Wdnm1-like, situated upstream of the gene HEAT Repeat Containing 6 (HEATR6) like its ortholog in eutherian mammals. To avoid gaps in the bulk of the figure, the N-terminus of the opossum sequence is not perfectly aligned with Wdnm1-like of eutherian mammals.\n\nExcept for rabbit (see Methods section), the figure shows the ORFs of sequences corresponding to the murine Wdnm1-like protein coding transcript of NCBI accession NM_183249, while other (possible) splicoforms are neglected. The intron site is indicated by a downward triangle. Intron sequences are not shown, but the below listed genomic sequence reports agree with GT-AG borders. For most of the species, the depicted sequences were supported by transcript reports, as exemplified per species in the Methods section. In the figure, dashes indicate gaps that were introduced for optimal sequence alignment. The alignments were performed by hand.\n\nAmino acid sequences are indicated above the second nucleotides of codons. Basic residues are indicated in red, acidic residues in blue, and green residues are more hydrophilic than the orange ones (following reference9). Cysteines are in violet. Asterisks correspond with stop codons. Predicted leader sequences are underlined.\n\nThe mouse Wdnm1-like sequence was designated “mouse lnc-DC ortholog” by Wang et al.1, and they targeted the regions shaded blue and green for transcript knockdown by “RNAi-1” and “RNAi-2”, respectively, using a lentivirus-mediated RNA interference system.\n\nThe name Wdnm1-like was first coined by Adachi et al.5, who found that Wdnm1-like transcript was differentially expressed in limbal versus central corneal epithelia in rat, and who observed similarity of the encoded protein with Wdnm1. Within the serial analysis of gene expression (SAGE) experiment by Adachi et al.5, Wdnm1-like comprised the most abundant SAGE tag present exclusively in the limbal library, and the authors hypothesized that Wdnm1-like might be a marker of limbal stem cells. They could, however, not rule out the possibility that Wdnm1-like was expressed by other cell types present in limbal epithelia, such as for example dendritic cells. A later study on rodent Wdnm1-like was performed in mice by Wu and Smas2. Wu and Smas got interested in Wdnm1-like after they found it highly upregulated upon differentiation of preadipocytes into adipocytes. They found Wdnm1-like to be selectively expressed in liver and adipose tissue, and enriched in white adipose depots versus brown. Recombinant expression of tagged murine Wdnm1-like in HT1080 human fibrosarcoma cells revealed a small secreted protein2. Because Wdnm1-like is a distant member of the whey acidic protein/four-disulfide core (WAP/4-DSC) family, of which several members have roles as proteinase inhibitors, Wu and Smas speculated that Wdnm1-like might have a similar function. An important class of extracellular proteases involved in adipocyte differentiation are the matrix metalloproteinases (MMPs), which can degrade extracellular matrix (ECM) components. Therefore, Wu and Smas investigated whether MMPs expressed by HT1080 were affected by the recombinant Wdnm1-like expression, and they found an increased amount of the active form of MMP-22. Thus, rather than having an inhibitory effect, Wdnm1-like appears to enhance activation of a protease. Wu and Smas conclude with “Future studies are required to address the mechanism(s) underlying the function and regulation of adipocyte-secreted Wdnm1-like”2, and according to literature this situation has not changed since then.\n\nLooking at the combined publications, a very complicated picture emerges. In most mammals the Wdnm1-like locus encodes a protein, with humans as an exception which is possibly unique. In rat, Wdnm1-like is differentially expressed in limbal versus central corneal epithelia4. In mouse, Wdnm1-like is expressed upon adipogenesis, and Wdnm1-like protein enhances the production of active MMP-22. In human and mouse, the Wdnm1-like(-ψ) transcript appears functionally associated with dendritic cell differentiation, and at least in humans this may be mediated by binding of the transcript to STAT31. This leaves questions for future research such as, for example, whether human Wdnm1-like-ψ transcript is also associated with adipogenesis, and whether murine Wdnm1-like transcript exerts its function on DC differentiation by binding to STAT3 or by encoding Wdnm1-like protein. Supporting that the Wdnm1-like proteins and transcripts in some extinct or extant animals may have (had) synergetic functions, is the fact that differentiation of both adipocytes and limbal epithelial cells can involve STAT36,7. So, despite our points of criticism, we think that the results and human model by Wang et al. may be valid and part of a more complex evolutionary scenario that involves distinct functions at the transcript and protein level, and a number of different tissues and cell types. In general, we think that studies on long noncoding RNAs typically require discussion of the evolutionary context8, especially when dealing with wide species borders such as between human and mouse.\n\nA nice speculation allowed by the combined referenced articles is that Wdnm1-like protein might promote Mo-DC differentiation in humans. After all, murine Wdnm1-like protein was found to enhance MMP-2 activity of human HT1080 cells2, concluding that humans did not lose their sensitivity to Wdnm1-like protein. The predicted mature Wdnm1-like protein is so small that it can be rapidly and commercially synthesized, enhancing the speed of possible investigation.\n\nWe did not feel comfortable with the amount of space and visibility the editors of the journal Science were able to offer us via their commenting mechanism for the discussion presented here. Therefore, we declined their offer, and instead deemed publication in F1000Research a more appropriate vehicle. Through F1000Research we hope to ask the corresponding authors of several of the referenced articles, including the article by Wang et al., to provide referee reports (which may also include broad views) on our discussion. We hope that this will lead to further discussions of our article. Since Wang et al. have the experimental system at hand, it might also be interesting if now or in the near future they could test whether (gorilla sequence?) Wdnm1-like protein can promote human Mo-DC differentiation.\n\nWdnm1-like protein appears to be very interesting. Not only may it be involved in differentiation of several cell types, it also is intriguing because it appears highly conserved throughout eutherian mammals and (rather) uniquely lost in hominids. It may help determine what makes us human.\n\n\nMethods\n\nThe partial Wdnm1-like sequence information available for extinct hominids, namely, for Neanderthal and Denisovan, was retrieved using the UCSC genome browser (http://genome.ucsc.edu). All other sequences shown in the figure were retrieved from Ensembl (www.ensembl.org/) or NCBI (http://www.ncbi.nlm.nih.gov/) databases. For a representative list of model species, we investigated database sequences of all mammals for which genomic sequences are available in the Ensembl database, and also of Pan paniscus (bonobo). For some of those animals sequence information for the Wdnm1-like ORF or for its expected genomic site was incomplete, and in such case the sequence is not included in the alignment figure.\n\nLeader peptide sequences were predicted by SignalP software (www.cbs.dtu.dk/services/SignalP/) and are underlined. For the species Panio anubis (olive baboon), Heterocephalus glaber (naked mole-rat), Myotis lucifugus (microbat), and Dasypus novemcintus (armadillo), besides the here indicated evolutionary conserved cleavage site, the SignalP software also predicted an alternative cleavage site with a calculated higher likelihood (not shown).\n\nSpecies-specific information related to sequences depicted in Figure 1:\n\nThe depicted human Wdnm1-like pseudogene sequence maps within Ensembl database GRCh37, Chr.17 positions 58162470-to-58165647, reverse orientation. Furthermore, the depicted sequence corresponds with positions 182-to-366 of the transcript sequence of NCBI accession NR_030732.1.\n\nThe depicted Neanderthal sequence was identified from genomic DNA fragments (Read names: M_SL-XAT_0004_FC30PMDAAXX:1:87:384:343, M_BIOLAB29_Run_PE51_1:2:9:981:262 and C_M_SOLEXA-GA04_JK_PE_SL21:8:99:944:526) isolated from the Vi33.16 and Vi33.25 Neanderthal samples10 using the UCSC genome browser by comparison with the human Wdnm1-like sequence. The depicted sequence fragment is identical to that of human.\n\nThe depicted Denisovan sequence was obtained as described for the Neanderthal sequences, and corresponds to part of the read M_SOLEXA-GA02_00040_PEdi_MM_3:8:112:19220:10730#AACCATG,CTCGATG (Meyer et al.11). The depicted sequence fragment is identical to that of human.\n\nThe depicted chimpanzee Wdnm1-like sequence maps within Ensembl database CHIMP2.1.4, Chr.17 positions 588018940-to-58805072, reverse orientation. Transcription is supported by NCBI SRA database sequence reports, such as for example gnl\|SRA\|DRR003370.54864751.1 of experiment set DRX002694.\n\nThe depicted bonobo Wdnm1-like sequence maps within the genomic sequence of NCBI database accession gb\|AJFE01016111.1\|, positions 11414-to-14585, forward orientation. Transcription is supported by NCBI SRA database sequence reports, such as for example gnl\|SRA\|SRR873628.59588401.2 of experiment set SRX290737.\n\nThe depicted gorilla Wdnm1-like sequence maps within Ensembl database gorGor3.1, Chr.5 positions 23775798-to-23778981, forward orientation. Transcription is supported by NCBI SRA database sequence reports, such as for example gnl\|SRA\|SRR306801.5146816.1 of experiment set SRX081945.\n\nThe depicted orangutan Wdnm1-like sequence maps within Ensembl database PPYG2, Chr. 17 32711864-to-32715478, forward orientation. This is a recent intrachromosomal duplication of the original Wdnm1-like gene. The Ensembl database shows that Sumatran orangutan still has at least part of that original Wdnm1-like gene upstream of HEATR6, but information of that region is incomplete. Evidence for transcription of Wdnm1-like in orangutan is provided by NCBI SRA database sequence reports for Pongo pygmaeus (Bornean orangutan), such as for example gnl\|SRA\|SRR306799.12707499.1 of experiment set SRX081943.\n\nThe depicted gibbon Wdnm1-like sequence maps within the genomic sequence of NCBI database accession gb\|ADFV01146912.1\|, positions 1414-to-4561, reverse orientation.\n\nThe depicted olive Wdnm1-like sequence maps within Ensembl database Panu_2.0, scaffold JH685681 positions 60156-to-64601, reverse orientation. Transcription is supported by NCBI SRA database sequence reports, such as for example gnl\|SRA\|SRR1045089.118535973.1 of experiment set SRR1045089.\n\nThe depicted rhesus macaque Wdnm1-like sequence maps within Ensembl database MMUL_1, scaffold 1099548049739 positions 121534-to-124737, forward orientation. Transcription is supported by NCBI SRA database sequence reports, such as for example gnl\|SRA\|SRR1240160.28991243.2 of experiment set SRR1240160.\n\nThe depicted crab-eating macaque Wdnm1-like sequence maps within the genomic sequence of NCBI database accession gb\|AEHL01027073.1\|, positions 5255-to-8524, forward orientation. Transcription is supported by NCBI SRA database sequence reports, such as for example gnl\|SRA\|DRR001354.3296367.1 of experiment set DRX000951.\n\nThe depicted Wdnm1-like sequence maps within Ensembl database ChlSab1.0, Chr.16 positions 29807079-to-29810262, reverse orientation. Transcription is supported by NCBI SRA database sequence reports for the closely related species Chlorocebus aethiops (green monkey), such as for example gnl\|SRA\|SRR1178509.592424.2 of experiment set SRR1178509.\n\nThe depicted squirrel monkey Wdnm1-like sequence maps within Ensembl database SalBol1.0, scaffold JH378137 positions 636410-to-639575, forward orientation. Transcription is supported by NCBI SRA database sequence reports, such as for example gnl\|SRA\|SRR500949.3269772.2 of experiment set SRX149650.\n\nThe depicted marmoset Wdnm1-like sequence maps within Ensembl database C_jacchus3.2.1, Chr.5 positions 88345809-to-88348914, reverse orientation. Transcription is supported by NCBI database accession gb\|GAMR01043615.1\|.\n\nThe depicted tarsier Wdnm1-like sequence maps within Ensembl database tarSyr1, scaffold_1716 positions 51738-to-55873, reverse orientation.\n\nThe depicted bushbaby Wdnm1-like sequence maps within Ensembl database OtoGar3, scaffold GL873613 positions 7509108-to-7514627, reverse orientation.\n\nThe depicted mouse lemur Wdnm1-like sequence maps within Ensembl database micMur1, GeneScaffold_1067 positions 49762-to-53887, reverse orientation. Transcription is supported by NCBI SRA database sequence reports, such as for example gnl\|SRA\|SRR832933.720157201.1 of experiment set SRX270644.\n\nThe depicted Chinese tree shrew Wdnm1-like sequence maps within Ensembl database TREESHREW, scaffold_15853 positions 2941-to-6216, reverse orientation. Transcription is supported by NCBI SRA database sequence reports, such as for example gnl\|SRA\|SRR518934.53798716.1 of experiment set SRX157966.\n\nThe depicted mouse Wdnm1-like sequence maps within Ensembl database GRCm38, Chr.11 positions 83747027-to-83749327, forward orientation. Transcription is supported by for example NCBI accession NM_183249.1.\n\nThe depicted rat Wdnm1-like sequence maps within Ensembl database Rnor_5.0, Chr.10 positions 70671110-to-70673427, forward orientation. Transcription is supported by for example NCBI accession gb\|EF122001.1\|.\n\nThe depicted prairie vole Wdnm1-like sequence maps within Ensembl database MicOch1.0, Chr.7 positions 15310620-to-15312860, reverse orientation. According to the Ensembl database the prairie vole also has an intronless copy of Wdnm1-like gene on Chr.X (not shown). Transcription is supported by NCBI SRA database sequence reports, such as for example gnl\|SRA\|SRR058428.108679.2 of experiment set SRX018513.\n\nThe depicted hamster Wdnm1-like sequence maps within the genomic sequence of NCBI database accession gb\|AMDS01007412.1\|, positions 15363-to-17750, forward orientation.\n\nThe depicted kangaroo rat Wdnm1-like sequence maps within Ensembl database dipOrd1, scaffold_2778 positions 48464-to-52516, reverse orientation.\n\nThe depicted squirrel Wdnm1-like sequence maps within Ensembl database spetri2, scaffold JH393300 positions 533158-to-536139, forward orientation.\n\nThe depicted naked mole-rat Wdnm1-like sequence maps within Ensembl database HetGla_female_1.0, scaffold JH602188 positions 3555009-to-3557720, forward orientation.\n\nThe depicted guinea pig Wdnm1-like sequence maps within Ensembl database cavPor3, scaffold_32 positions 10788821-to-10791159, reverse orientation.\n\nThe depicted rabbit Wdnm1-like sequence maps within Ensembl database OryCun2.0, Chr.19 positions 25079843-to-25084775, forward orientation. The underlined part in Italic font at the 3’end belongs to a third exon. Transcription is supported by for example NCBI accession gb\|GBCH01008538.1\|.\n\nThe depicted pika Wdnm1-like sequence maps within Ensembl database OchPri3, scaffold JH802106 positions 113719-to-116807, forward orientation. Transcription is supported by NCBI SRA database sequence reports, such as for example gnl\|SRA\|SRR850200.108627.2 of experiment set SRX277346.\n\nThe depicted cattle Wdnm1-like sequence maps within Ensembl database UMD3.1, Chr.19 positions 14485956-to-14490393, reverse orientation. Transcription is supported by for example NCBI accession gb\|AW484602.1\|.\n\nThe depicted sheep Wdnm1-like sequence maps within Ensembl database Oar_v3.1, Chr.11 positions 13759463-to-13763966, reverse orientation. Transcription is supported by for example NCBI accession gb\|CK830678.1\|.\n\nThe depicted dolphin Wdnm1-like sequence maps within the genomic sequence of NCBI database accession gb\|ABRN02348024.1\|, positions 2742-to-5945, forward orientation.\n\nThe depicted pig Wdnm1-like sequence maps within the genomic sequence of NCBI database accession gb\|AJKK01193461.1\|, positions 3786-to-7100, reverse orientation. Transcription is supported by for example NCBI accession dbj\|AK399701.1\|.\n\nThe depicted alpaca Wdnm1-like sequence maps within Ensembl database vicPac1, GeneScaffold_1352 positions 716864-to-719675, reverse orientation.\n\nThe depicted horse Wdnm1-like sequence maps within Ensembl database EquCab2, Chr.11 positions 36986601-to-36989473, reverse orientation. The Ensembl database indicates additional copies of Wdnm1-like on Chr.11 (not shown). Transcription is supported by for example NCBI accession gb\|DN508620.1\|.\n\nThe depicted rhinoceros Wdnm1-like sequence maps within Ensembl database CerSimSim1, scaffold JH767772 positions 17445128-to-17447968, reverse orientation.\n\nThe depicted microbat Wdnm1-like sequence maps within Ensembl database Myoluc2.0, scaffold_GL430154 positions 92198-to-94525, reverse orientation.\n\nTranscription is supported by NCBI SRA database sequence reports, such as for example gnl\|SRA\|SRR1013468.27145136.1 of experiment set SRR1013468.\n\nThe depicted cat Wdnm1-like sequence maps within the genomic sequence of NCBI database accession gb\|AANG02057756.1\|, positions 9507-to-13123, forward orientation. Transcription is supported by NCBI SRA database sequence reports, such as for example gnl\|SRA\|SRR835496.27404932.1 of experiment set SRX272142.\n\nThe depicted dog Wdnm1-like sequence maps within Ensembl database CanFam3.1, Chr.9 positions 37619501-to-37622242, reverse orientation. Transcription is supported by for example NCBI accession gb\|DR107055.1\|.\n\nThe depicted panda Wdnm1-like sequence maps within Ensembl database ailMel1, scaffold GL193203 positions 54404-to-57462, forward orientation.\n\nThe depicted ferret Wdnm1-like sequence maps within Ensembl database MusPutFur1.0, scaffold GL896917 positions 9435086-to-9438171, forward orientation. Transcription is supported by for example NCBI accession gb\|JR792458.1\|.\n\nThe depicted tenrec Wdnm1-like sequence maps within the genomic sequence of NCBI database accession gb\|AAIY02150441.1\|, positions 1061-to-5393, forward orientation.\n\nThe depicted elephant Wdnm1-like sequence maps within Ensembl database loxAfr3, scaffold_31 positions 5685863-to-5689773, forward orientation. Transcription is supported by NCBI SRA database sequence reports, such as for example gnl\|SRA\|SRR1041765.37646273.1 of experiment set SRR1041765.\n\nThe depicted aardvark Wdnm1-like sequence maps within Ensembl database OryAfe1, scaffold JH863914 positions 5948889-to-5951515, reverse orientation.\n\nThe depicted armadillo Wdnm1-like sequence maps within Ensembl database Dasnov3.0, scaffold JH562945 positions 1888971-to-1892017, forward orientation. Transcription is supported by NCBI SRA database sequence reports, such as for example gnl\|SRA\|SRR494776.6845635.2 of experiment set SRX146634.\n\nThe depicted opossum Wdnm1-like sequence maps within Ensembl database BROADO5, Chr.2 positions 498828348-to-498830609, reverse orientation. Transcription is supported by NCBI SRA database sequence reports, such as for example gnl\|SRA\|SRR908062.57922637.2, gnl\|SRA\|SRR873400.62402918.1, gnl\|SRA\|SRR943348.21681365, gnl\|SRA\|SRR943348.11424624, and gnl\|SRA\|SRR943348.9801988 of experiment sets SRX310006 and SRX290643 (because other Wdnm1-like transcript information appears lacking for marsupials, we here provide SRA database accessions that together cover the full ORF).", "appendix": "Author contributions\n\n\n\nJMD did most of the research and wrote the manuscript. KTB analyzed sequence databases of extinct hominids and carefully checked the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in the funding of this work.\n\n\nReferences\n\nWang P, Xue Y, Han Y, et al.: The STAT3-binding long noncoding RNA lnc-DC controls human dendritic cell differentiation. Science. 2014; 344(6181): 310–3. PubMed Abstract \| Publisher Full Text\n\nWu Y, Smas CM: Wdnm1-like, a new adipokine with a role in MMP-2 activation. Am J Physiol Endocrinol Metab. 2008; 295(1): E205–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJackson AL, Burchard J, Schelter J, et al.: Widespread siRNA “off-target” transcript silencing mediated by seed region sequence complementarity. RNA. 2006; 12(7): 1179–87. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKenworthy R, Lambert D, Yang F, et al.: Short-hairpin RNAs delivered by lentiviral vector transduction trigger RIG-I-mediated IFN activation. Nucleic Acids Res. 2009; 37(19): 6587–99. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAdachi W, Ulanovsky H, Li Y, et al.: Serial analysis of gene expression (SAGE) in the rat limbal and central corneal epithelium. Invest Ophthalmol Vis Sci. 2006; 47(9): 3801–10. PubMed Abstract \| Publisher Full Text\n\nDerecka M, Gornicka A, Koralov SB, et al.: Tyk2 and Stat3 regulate brown adipose tissue differentiation and obesity. Cell Metab. 2012; 16(6): 814–24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHsueh YJ, Chen HC, Chu WK, et al.: STAT3 regulates the proliferation and differentiation of rabbit limbal epithelial cells via a ΔNp63-dependent mechanism. Invest Ophthalmol Vis Sci. 2011; 52(7): 4685–93. PubMed Abstract \| Publisher Full Text\n\nNecsulea A, Soumillon M, Warnefors M, et al.: The evolution of lncRNA repertoires and expression patterns in tetrapods. Nature. 2014; 505(7485): 635–40. PubMed Abstract \| Publisher Full Text\n\nHopp TP, Woods KR: Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci U S A. 1981; 78(6): 3824–28. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGreen RE, Krause J, Briggs AW, et al.: A Draft Sequence of the Neandertal genome. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "5905", "date": "21 Aug 2014", "name": "Cynthia Smas", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis laboratory (Smas) published a manuscript on the Wdnm1-like murine gene in 20081; as such we have been invited to review and comment here on the paper by Dijkstra and Ballingall. Their report presents extensive sequence analysis, for a range of species, of the Wdnm1-like gene. The title of the report is an accurate representation of the content of the article and the important points are well-summarized in the abstract. The extensive sequence information they provide in their text is in a format that allows the reader to reassess the sequence analysis data if so desired.The information provided by Dijkstra and Ballingall makes it abundantly clear that the Wdnm1-like locus is predicted to contain a bona fide open reading frame that would encode a small secreted protein in all extant species examined except humans. This protein is Wdnm1-like, a member of the WFDC (Whey Acidic Protein Four Disulfide Core) protein family. In all but hominids, the predicted protein encoded by the Wdnm1-like gene, also recently annotated in the Gene NCBI database as Wfdc21, has clear homology with the WFDC protein family2. In hominids (Homo sapiens) this locus contains the Wdnm1-like pseudogene, which Wang and co-authors report encodes a long non-coding RNA that they have named lnc-DC. In humans, a one base pair deletion is present near the start of what would have been the open reading frame for the Wdnm1-like protein. This frame shift apparently eliminates the protein coding ability of the human gene. However, restoration of the correct open reading frame would be predicted to generate an encoded protein with significant homology to the murine Wdnm1-like protein.The detailed sequence analyses and other information provided by Dijkstra and Ballingall is valuable to the long-noncoding RNA research community and to those following the recent research into dendritic cell differentiation, in respect to the Science report that was published in April 2014 by Wang and co-authors “The STAT3-Binding Long Noncoding RNA lnc-DC Controls Human Dendritic Cell Differentiation” 3. In regard to the Science report by Wang and co-authors the design, methods and analysis of the results in the paper by Dijkstra and Ballingall are well-explained and are they appropriate to the topic. The conclusions they reach are sensible, generally balanced and justified. In a few instances, however, the tone of the writing is a bit aggressive. As further discussed below, it appears an assumption is made by Dijkstra and Ballingall that Wang and co-authors were aware of the distinction that the human locus encodes a pseudogene, while in mouse this locus encodes the Wdnm1-like protein.This laboratory (Smas) identified murine Wdnm1-like as an adipogenesis-induced gene in a report in 2008, wherein a role for Wdnm1-like protein in modulating MMP activity was also reported. Shortly after this publication in 2008, when considering generation and study of null mice for Wdnm1-like, it became apparent that the human locus had a single base pair deletion early in an otherwise predicted open reading frame. Therefore, it likely encoded a pseudogene in humans and not the Wdnm1-like protein. Given this, it was decided that this laboratory (Smas) would not go further on the project in respect to the study of the Wdnm1-like protein. As such, the human gene or transcript has not been addressed in studies from this laboratory (Smas), and we not aware of any data reporting on expression of this transcript in human adipose tissue. However, while not the subject of such studies, murine Wdnm1-like has been mentioned within several publications on adipocytes and adipose tissue 4-6. These relate to the finding that the Wdnm1-like transcript is highly enriched in expression in white vs. brown murine adipocytes/adipose tissue 4-6. Of interest, knockout mice for Wfdc21 (Wfdc21tm1a(KOMP)Wtsi ) are now available by embryo resuscitation through the KOMP project (Project ID: CSD49368) and would serve as a highly useful system in which to address the role of Wdnm1-like in DC cell maturation in mice. In the prior publication on Wdnm1-like from this laboratory (Smas), our studies utilized a murine Wdnm1-like expression construct with a C-terminal epitope tag, and cell transfection studies. These showed that a protein of predicted size for Wdnm1-like was ectopically expressed and secreted into culture media. Dijkstra and Ballingall do accurately describe our studies and clearly state that our work on Wdnm1-like utilized recombinant ectopic expression of the predicted murine Wdnm1-like open reading frame. However, one would have liked to have seen Dijkstra and Ballingall make it even more clear, earlier on in their text, that the endogenous Wdnm1-like protein has not yet been demonstrated in any system/species. A quick Google search fails to find an available antibody to the Wdnm1-like protein, so the endogenous protein remains to be investigated. The work by Dijkstra and Ballingall makes several very important points: It provides well-needed clarification and extensive documentation that apparently only for hominids does the locus for Wdnm1-like encode a long non-coding RNA, while in all other species specifically examined (which was quite extensive) this locus contains an open reading frame for the Wdnm1-like protein. It raises concerns as to the ultimate responsibility of Wang and co-authors to report the full range of information on such distinctions within their Science report, if they indeed were aware of such. Only Wang and co-authors can inform us of their extent of knowledge of the protein coding nature of murine vs. human Wdnm1-like at the time of their Science publication. Thus it is not possible at this point to know whether such information was selectively omitted. But as Dijkstra and Ballingall point out, Wang and co-authors refer to the GenBank entry for murine Wdnm1-like , also known as 100001G20Rik and now formally named Wfdc21. This GenBank entry contains citations for publications on murine Wdnm1-like 1,7. It seems very odd if Wang and co-authors were not aware of the distinctions between the human and murine forms of Wdnm1-like, particularly in today’s age of well-curated databases. In fact, the NCBI Unigene entry for this gene reveals the human version is annotated as Wdnm1-like pseudogene (LOC645638, Hs.463652). Perhaps this was one more instance of a research group “rediscovering and renaming” a gene that was previously published on. This is all too common of late; doing so essentially tosses aside the already peer-reviewed and published work of others 1,7. However, until further clarification is forthcoming by Wang and co-authors, one would hope they would be provided with the benefit of the doubt on the facts and intentions in regard to this matter. A third valid concern raised by Dijkstra and Ballingall, is whether the knowledge that the murine gene is predicted to be protein encoding, while the human gene encodes a long non-coding RNA (lnc-DC), impacts the quality or interpretation of the data in the manuscript by Wang and co-authors. It appears that for the vast majority of the studies in their Science publication, Wang and co-authors utilized human cell culture systems, and limited studies were conducted in murine systems. Thus we are in agreement with Dijkstra and Ballingall that the essential conclusions of the Science report are not dependent on the murine studies, or the fact that Wdnm1-like encodes a long non-coding RNA in humans and the Wdnm1-like protein in mice. However Wang and co-authors used studies in murine systems to further address whether knockdown of Wdnm1-like affected DC differentiation, finally claiming “that lnc-DC is vital for DC differentiation in both human and mice”. This strikes one as a very disturbing claim as it implies that in mice this gene locus functions as a long non-coding RNA, when all the available evidence clearly argues against such. This leaves the readers of Science with less than the complete and indeed even an obfuscated picture of the Wdnm1-like gene. Readers, and one presumes also the reviewers of the Science report, were thus unable to fully judge the quality and relevance of the studies that Wang and co-authors conducted in murine systems. As indicated by Dijkstra and Ballingall, Wang and co-authors referring to or renaming murine Wdnm1-like as the “mouse lnc-DC ortholog”, without also educating their readers on the protein coding nature of the murine gene, appears a serious disservice to their readers. If Wang and co-authors were aware of the fact that murine Wdnm1-like gene was most likely protein-encoding, and whether this was the case or not is unknown at this juncture, they have indeed failed the readers of Science in this regard. One would hope to see some explanation from Wang and co-authors on this matter. The points raised by Dijkstra and Ballingall serve as “food for thought” for all of us on the responsibility of authors to fully inform their readers in regard to the state of current knowledge in respect to the scientific content of their manuscripts. It is left up to the readers of the paper by Dijkstra and Ballingall, the text of the Science report by Wang and co-authors and the comments furnished herein, to reach their individual opinions on this specific matter.", "responses": [ { "c_id": "952", "date": "27 Aug 2014", "name": "Johannes M. Dijkstra", "role": "Author Response", "response": "Dear Dr. Smas and Dr. Ren,Thank you for your extensive comments, which we embrace as generally positive. We are happy that you approve of how we summarized existing reports on Wdnm1-like in rodents. Furthermore, you added some nice insights and discussion points, and provided the readers with additional references that confirm that murine Wdnm1-like transcript is highly enriched in white vs. brown adipocytes/adipose tissue. You state correctly that endogenous Wdnm1-like protein has not been reported so far. However, a search of the PeptideAtlas database http://www.peptideatlas.org (Desiere, et al. 2006) which includes peptides identified by mass spectrometry from multiple species, identified a rat peptide encoded by correctly spliced Wdnm1-like . This suggests that the Wdnm1-like protein is present in the rat. We will include this information in the text after all the referee reports have been received. Whether the style of our manuscript is excessively critical is open to discussion, especially since both our research groups appear to share similar criticisms of the Wang et al. report. However, if you feel that a sentence is excessively critical please send us suggestions for changes (in a private mail?) and we will follow your lead.Sincerely,Johannes M. Dijkstra and Keith T. BallingallReferenceDesiere, et al., \"The PeptideAtlas Project\", Nucleic Acids Research, 2006, 34, D655-D658" } ] }, { "id": "6091", "date": "09 Sep 2014", "name": "Julja Burchard", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis item of correspondence is in reference to Wang et al. (2014), concerning possible cytosolic protein-binding function in conventional dendritic cells by a human noncoding RNA and its mouse ortholog. The correspondents note that the lncRNA designated “lnc-DC” by Wang et al. is a protein-coding transcript, Wfdc21 or Wdnm1-like, in all mammals with available sequence other than hominids (Homo sapiens sapiens, Neanderthal and Denisova), and suggest that Wang et al. would have been well advised to discuss this information, as they propose functions for the transcript in human and mouse cells.The correspondent authors provide an abbreviated summary of the original article in the abstract with suitable detail in the body of the letter. The letter brings up an intriguing point worthy of discussion. Drs. Dijkstra and Ballingall frame and present their observations well and do a thorough job assembling and aligning sequences in support. To their point, recent work finds that only 0.3% of anthropoid-specific constrained sequences – functional primate innovations – are coding (del Rosario et al., 2014). If a similar proportion holds for significant functions hominids have ceased to perform, Wang et al. have identified an unusual case and have not highlighted it as such. A few items of follow-up may be of interest to clarify and extend the evolutionary observations in this correspondence.Is the frameshift in the erstwhile signal peptide coding sequence in the human reference genome reproduced in all 1000-genomes data, or is WFDC21P a polymorphic human pseudogene? Is guidance by the human reference sequence in assembly of Neanderthal and Denisova genomes a potential factor in their reproduction of the reported human frameshift? Is there human genetic variation at this locus tied to variation in trait expression, or alternatively is there evidence that variation in this small gene is sufficiently suppressed to leave no functional variation for genetic association studies to mine? Either could be consistent with a significant role in immune function as proposed by Wang et al. What is found at the position syntenic to Wfdc21 in marsupials other than the one noted as sharing this gene, and in lower model organisms?\n\nThis may help clarify the nature of the apparent mammalian innovation at this locus. Do regulatory elements for WFDC21P differ between hominids and other species? If Wfdc21 is a gene with active RNA and protein products, their function will have evolved in the context of cell-type specific expression. Fantom5 CAGE tag data suggests a difference in regulation of human WFDC21P vs. mouse Wfdc21. Although parallel samples are not available for all tissues and cell types, mouse data show strongest expression in myeloid suppressor cells with significant expression also in liver and skin, while human data show 1000x lower maximum expression with best expression in migratory Langerhans cells. Do the sections of WFDC21P-RNA highlighted as functional by Wang et al. show signs of hominid or mammalian constraint? It is intriguing that Wang et al. show data suggesting STAT3 binding by the 3’-end of WFC21P-RNA in a section downstream of the ORF and thus potentially available for secondary function. The correspondent authors also discuss the technical merits of the work presented in Wang et al., as indicated by the potential novelty of the findings. Four additional points may be made here.It is important to establish the RNA-dependence of activities discussed by Wang et al. While the correspondents are satisfied that lack of human protein coding has been demonstrated, some experiments could be clearer. Wfdc21 is a secreted protein. Did Wang et al. examine the supernatant as well as the cells in which Flag-tagged fusions were expressed? The correspondents comment on the inconsistent use of controls. Indeed, the sole controlled experiment suggesting RNA-protein association appears to be the pulldown of STAT3 with biotinylated WFDC21P RNA, with specific absence of the STAT3 band in the antisense control. While RIP with STAT3 experimental and STAT1 control antibodies was conducted, no sequencing is reported so the specificity of interaction with STAT3 is not known. Further, figures on RNA-FISH visualization of association of STAT3p with WFDC21P-RNA do not show controls. Wang et al. rely on inhibitors to demonstrate WFDC21P-RNA function. As the correspondents note, one shRNA sequence is primarily employed and it is not consistently paired with varied on- or off-target controls. Literature on functional siRNA screens suggests that a half dozen RNAi sequences with independent seeds are required for dissociation of off- and on-target activities. Further, Wang et al. have performed expression profiling on shRNA-treated cells. The profiles can be examined for seed-based off-target activity and for inflammatory response to the lentiviral vector according to published methods. It will be important to establish whether the dendritic cell proteins whose differential expression is highlighted by Wang et al. show shRNA-matching seed sequences in their 3’UTRs or respond to lentiviral infection. Wang et al. also use published STAT3 inhibitors to elucidate the role of WFDC21P-RNA. It would be intriguing to speculate that an RNA-protein interaction site helps to define the STAT3 binding site of published inhibitor S3I-201 and its effects on STAT3 activity. However, the supplementary material provided by Wang et al. show much more profound effects on cytokine production by small molecules than shown for WFDC21P shRNA in the main paper, although effects on T-cell activation remain similar.", "responses": [ { "c_id": "970", "date": "10 Sep 2014", "name": "Johannes M. Dijkstra", "role": "Author Response", "response": "Dear Dr. Burchard, We thank you for your extensive and valuable comments. Like the comments by Drs. Smas and Ren, we embrace them as generally positive. Your comments add accuracy to our story, especially regarding the technical part of the RNA investigation by Wang and co-workers.You wonder, as do Drs. Smas and Ren, and as do we, whether in some individuals or under some conditions, humans may express Wdnm1-like protein. After all, the sequence for the mature protein appears intact, and requirements for a leader peptide are not very unique. However, our rather extensive database investigations could not retrieve a human sequence expected to encode a functional Wdnm1-like protein. Identical frameshifts in Neanderthal and Denisovan Wdnm1-like sequence reports, for the reliability of which we have to depend on the respective authors, argue against the likeliness of functional/nonfunctional Wdnm1-like polymorphism in modern humans. An expressed sequence tag (EST), reported as GenBank accession CD692402, suggests that an individual from southern China may have a protein coding Wdnm1-like sequence; however, besides repair of the frame-shift in the leader coding region, this sequence has an additional unique modification, and the sequence report may contain technical errors. In short, we could not obtain evidence for intact Wdnm1-like coding sequences in humans, but cannot exclude the possibility that such sequences exist. We would welcome if anyone could provide such evidence or indications. We took a brief look at Wdnm1-like evolution beyond eutherian mammals. However, except for the mentioned case in opossum, at this evolutionary distance it becomes difficult to distinguish Wdnm1-like orthologues from other family members, and it would become more a discussion on the evolution of Wdnm1-like plus related molecules than of Wdnm1-like alone. Because the primary goal of our study is the discussion of the Wang et al. article, which is confined to eutherian mammals, we feel a discussion of deeper Wdnm1-like evolution would make our study too complicated.Although we did not make systematic comparisons among various genes, we feel that overall the 3’-end region of human Wdnm1-like-y transcript which is believed to interact with STAT3, is not especially well conserved among mammals. However, without knowing the precise sequence motif or RNA secondary structure important for that binding, we probably shouldn’t speculate on presence or absence of evolutionary constraints on that region.You raise four additional points regarding the technical merits of the work presented by Wang and co-workers. Importantly, you agree with us that the Wang et al. study was inconsistent and probably incomplete in the use of controls. Some points you raise are valid speculations and questions, whereas others can be considered as criticism of the Wang et al. article. In our opinion that criticism is mostly right. However, we prefer not to change the open style of our technical comments, and hope that the readers will find the specific issues that you raise when reading your report.Thank you again for your hard and valuable work. Sincerely, Johannes M. Dijkstra and Keith T. Ballingall" } ] } ]	1	https://f1000research.com/articles/3-160
https://f1000research.com/articles/3-229/v1	29 Sep 14	{ "type": "Correspondence", "title": "[18F]-T807 tauopathy PET imaging in chronic traumatic encephalopathy", "authors": [ "Sam Gandy", "Steven T. DeKosky", "Steven T. DeKosky" ], "abstract": "A new molecular ligand for positron emission tomography (PET) of the human brain, [18F]-T807, is under investigation for the antemortem detection of pathological neurofibrillary aggregates, which are evidence of neurofibrillary tangle (NFT) diseases, also known as tauopathies. Repetitive mild traumatic brain injuries in athletes and battlefield veterans are associated with one such tauopathy, known as chronic traumatic encephalopathy (CTE). In a recent case report, a former NFL player with clinically probable CTE and a concurrent Progressive Supranuclear Palsy (PSP) –like syndrome was studied using [18F]-T807. The interpretation of this player’s [18F]-T807 PET imaging was complicated by the overlap of tracer uptake in brain regions involved in CTE and PSP with regions associated with either nonspecific [18F]-T807 ligand binding or “aging-associated” binding of [18F]-T807 to authentic tauopathy known to be associated with aging and disease severity (i.e., NFT in the mesial temporal lobe). The implications of these data for the utility of [18F]-T807 in the pre-mortem detection of CTE are summarized.", "keywords": [ "We recently reported the case of a retired NFL player with a clinical diagnosis of chronic traumatic encephalopathy (CTE)", "the diagnosis was based on clinical characteristics of his progressive cognitive disorder and negative [18F]-florbetapir PET brain imaging indicating absence of amyloid plaques in his brain (Mitsis et al. 2014)1. An [18F]-T807 PET imaging study revealed retention of [18F]-T807 in the substantia nigra (SN)", "globus pallidi (GP)", "and hippocampi", "bilaterally. Key images from that paper were reproduced in a recent review by Gandy et al. (2014)2. The pattern of retention was interpreted by the original authors as potentially consistent with the clinical diagnosis of CTE1." ], "content": "Correspondence\n\nWe recently reported the case of a retired NFL player with a clinical diagnosis of chronic traumatic encephalopathy (CTE); the diagnosis was based on clinical characteristics of his progressive cognitive disorder and negative [18F]-florbetapir PET brain imaging indicating absence of amyloid plaques in his brain (Mitsis et al. 2014)1. An [18F]-T807 PET imaging study revealed retention of [18F]-T807 in the substantia nigra (SN), globus pallidi (GP), and hippocampi, bilaterally. Key images from that paper were reproduced in a recent review by Gandy et al. (2014)2. The pattern of retention was interpreted by the original authors as potentially consistent with the clinical diagnosis of CTE1.\n\nThe anatomical distribution of [18F]-T807 retention was interpreted as atypical and suggestive of the distribution of pathology in Progressive Supranuclear Palsy (PSP). That interpretation was noted in the Mitsis et al. (2014) paper1 in light of a recent case report linking CTE and PSP3 and because the subject in the Mitsis et al. (2014)1 case manifested nasal speech, hypomimia, and impaired upgaze, all features of PSP. These facts notwithstanding, we emphasize that we cannot exclude the possibility that the pattern of [18F]-T807 binding observed in the retired NFL player could be the result of nonspecific retention of [18F]-T807 in these same regions of brain. Indeed, some as-yet unpublished experience with [18F]-T807 has demonstrated a propensity of the ligand to bind to the SN and GP in what appears to be a non-specific fashion (http://www.alzforum.org/news/conference-coverage/tau-tracers-shine-boston-conference). The [18F]-T807 binding to the hippocampi in1 could fall within the spectrum of aging-related tauopathy, the known deposition of NFT in this region (http://www.alzforum.org/news/conference-coverage/tau-tracers-shine-boston-conference). We write here to emphasize the point that proof of the histological underpinnings of [18F]-T807 binding awaits the presentation of a sufficiently powered study of in vivo-radiological/postmortem-histological correlation relationships, and as a reminder that while the development of several putative tau-binding ligands has opened new possibilities for research and clinical use, that none have been validated to the extent necessary for reliable use and such validation should be a top priority.", "appendix": "Author contributions\n\n\n\nThis manuscript was drafted by S.G. and edited by S.T. DeK.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nMitsis EM, Riggio S, Kostakoglu L, et al.: Tauopathy PET and amyloid PET in the diagnosis of chronic traumatic encephalopathies: studies of a retired NFL player and of a man with FTD and a severe head injury. Transl Psychiatry. 2014; 4: e441. PubMed Abstract \| Publisher Full Text\n\nGandy S, Ikonomovic MD, Mitsis E, et al.: Chronic traumatic encephalopathy: clinical-biomarker correlations and current concepts in pathogenesis. Mol Neurodegener. 2014; 9(1): 37. PubMed Abstract \| Publisher Full Text\n\nLing H, Kara E, Revesz T, et al.: Concomitant progressive supranuclear palsy and chronic traumatic encephalopathy in a boxer. Acta Neuropathol Commun. 2014; 2: 24. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "6252", "date": "06 Oct 2014", "name": "Victor Villemagne", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis timely letter by Drs Gandy and DeKosky sheds light onto the inherent difficulties of using not yet fully characterized imaging markers where lack of validation of the tracers used, coupled with limited experience in an emergent field, might potentially lead to over- or under-interpretation of the results, especially when no controls are available to juxtapose the findings. This in turn might reflect badly on the value of this new approach. Larger cohort studies and, as the authors point out, neuropathological confirmation of the imaging findings will be required to fully validate and characterize the binding of these new and essential markers for the in vivo imaging of tau deposits in the brain.", "responses": [] }, { "id": "6684", "date": "20 Nov 2014", "name": "Nobuyuki Okamura", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this letter, the authors describe an alternative explanation of [18F]T807 PET findings in patients with chronic traumatic encephalopathy (CTE). A striking article by Mitsis et al. (2014) has suggested the potential usefulness of tau PET imaging for the early detection of tau deposits in patients with CTE. The authors of the letter state that tracer signals in the substantia nigra and globus pallidus might be the result of nonspecific retention of [18F]T807. However, the amount of hippocampal [18F]T807 retention (SUVR 1.45) was considerably higher than the values that have been previously reported in healthy controls, suggesting the involvement of the hippocampus in this patient. It is important to confirm whether the hippocampal [18F]T807 retention was within normal age-appropriate levels or whether it reflected CTE tau lesions in this case. Furthermore, the current findings should be confirmed by postmortem examinations in the future.", "responses": [] } ]	1	https://f1000research.com/articles/3-229
https://f1000research.com/articles/3-129/v1	17 Jun 14	{ "type": "Case Report", "title": "Case Report: Double lumen tube insertion in a morbidly obese patient through the non-channelled blade of the King Vision™ videolaryngoscope", "authors": [ "Mohamed El-Tahan", "D. John Doyle", "Alaa M Khidr", "Ahmed G Hassieb", "D. John Doyle", "Alaa M Khidr", "Ahmed G Hassieb" ], "abstract": "We describe the insertion of the double lumen endobronchial tube (DLT) using a non-channeled standard blade of the King VisionTM videolaryngoscope for one lung ventilation (OLV) in a morbidly obese patient with a predicted difficult airway, severe restrictive pulmonary function, asthma, and hypertension. The patient was scheduled for a video-assisted thoracoscopic lung biopsy. The stylet of the DLT was bent to fit the natural curve of the #3 non-channeled blade of the King Vision™ videolaryngoscope. We conclude that the use of King Vision™ videolaryngoscope could offer an effective method of DLT placement for OLV.", "keywords": [ "one lung ventilation", "difficult intubation", "double lumen tube", "King Vision™ videolaryngoscope", "thoracic surgery" ], "content": "Introduction\n\nThe GlideScope® (Verathon Inc., Bothell, WA, USA) has been used to facilitate the placement of the double lumen endobronchial tubes (DLT) in patients with a difficult airway1,2. However, DLT placement in patients with a limited mouth opening is relatively difficult compared to a single-lumen tube (SLT) because of the larger outer diameter, the distal curvature and the increased rigidity3,4. The DLT version of the channeled Airtraq® laryngoscopes (Prodol Limited, Viscaya, Spain) is equivalent in performance to direct laryngoscopy with a Macintosh blade4.\n\nThe King Vision™ video laryngoscope (King Systems, Indianapolis, IN, USA) is a portable video laryngoscope (VL) similar to the Pentax Airway Scope® (Pentax-AWS, Hoya Corp., Tokyo, Japan), but different in that the LED light and CMOS camera are part of the disposable blades. These blades are available in two styles: a standard non-channeled blade that requires the use of a stylet shaped to 60–70° to direct the SLT, and a channeled blade that incorporates a guide channel which directs the SLT towards the glottis. Both designs include an anti-fog lens coating. The height and width of the standard non-channeled and channeled blades are 13 mm and 26 mm vs. 18 mm and 29 mm, respectively. Among the Airtraq®, the Pentax Airway Scope® and the King Vision™ VL, the standard non-channeled blade of the King Vision™ VL has the smallest diameter.\n\nIn this report we show how the use of the standard non-channeled blade of the King Vision™ videolaryngoscope can be useful for DLT placement, as illustrated in the management of a morbidly obese patient with predicted difficult airway and severely restrictive pulmonary dysfunction.\n\n\nCase report\n\nA 52 year-old, 151 cm, 95 kg (body mass index 41.7 kg/m2) Asian woman presented with progressive orthopnea, dyspnea, and cough and was admitted to hospital. She had a 15 years history of moderate asthma, hypertension and hypocalcemia and was treated with irbesartan 150 mg/day, furosemide 40 mg/day, calcium carbonate 1.2 g/day and inhaled salbutamol.\n\nOn physical examination, dyspnea on mild exertion was present. The respiratory rate (RR) was 17/min, the resting heart rate (HR) was 80/min, blood pressure (BP) was 150/90 mm Hg and arterial oxygen saturation (SpO2) was 90% on a room air. Examination of the other systems (including abdomen and central nervous system examinations) revealed no abnormalities. Preoperative airway examination revealed a Mallampati class III airway, with an intercisor distance of 3.5 cm, a thyromental distance of 6.0 cm, normal teeth, and a full range of neck flexion and extension.\n\nChest radiography showed reticular opacities with honeycombing. Electrocardiography showed left axis deviation and poor R wave progression. Transthoracic echocardiography showed impaired left ventricular relaxation, mild apical wall hypokinesis and an ejection fraction of 0.55. The patient's electrolytes and creatinine were normal. Hemoglobin concentration was 12.9 g/dl and ionized calcium was 0.7 mmol/l. Pulmonary function testing showed a severe restrictive pattern (forced expiratory volume in first second [FEV1] 44.5%, forced vital capacity [FVC] 40.5%, and FEV1/FVC 109% of predicted). Arterial blood gases analysis showed hypoxemia on room air (pH 7.39, PaCO2 46.6 mmHg, HCO3 27.7 mmol/l, PaO2 58 mmHg).\n\nThe patient was scheduled for a video-assisted thoracoscopic lung biopsy. Multidisciplinary discussions involving a cardiothoracic surgeon, a pulmonologist, anesthesiologists and the family of the patients took place, emphasizing the possibility of acute pulmonary compromise during tracheal intubation and surgery. Awake fibreoptic intubation was offered as the best airway management option, but the patient refused. Written informed consent was obtained for tracheal intubation after induction of general anesthesia with the adopted stepwise plan.\n\nA stepwise plan was formulated: the initial plan included induction of general anesthesia through the placement of a left DLT using the King Vision™ VL. Backup plans were revised involving the insertion of the left DLT over a placed Eschmann tracheal tube introducer (Smiths-Medical International Ltd, Hythe, Kent, UK), and using a King Vision™ VL, an Arndt’s endobronchial blocker placed through a SLT. The use of selective lobar blockade was considered, if needed to correct hypoxemia during lung ventilation.\n\nOxygen at 3 L/min was delivered via a nasal cannula inserted upon entry of the patient in the operating room. Glycopyrrolate 0.2 mg was administered intravenously. Patient monitoring included electrocardiography, pulse oximetry, invasive arterial blood measurement, capnography, train of four stimulation of the ulnar nerve, and entropy-based depth of anesthesia monitoring. A left thoracic paravertebral catheter was inserted. No sedative premedication was given.\n\nAfter positioning of the patient on the operating table in a head-up position, anesthesia was induced using a target-controlled infusion (TCI) of sufentanil with a target effect site concentration (Ce) of 0.1 ng/mL, in conjunction with 8% sevoflurane in oxygen delivered by mask ventilation.\n\nLaryngoscopy was performed using a King Vision™ videolaryngoscope, where a grade II view of the glottis was observed. Succinylcholine (80 mg) was then administered intravenously for muscle relaxation.\n\nThe stylet of a 35 Fr left DLT (Portex® Blueline Endobronchial tube, Smiths Medical Intl. Ltd., Hythe, Kent) was bent to fit the natural curve of a standard non-channeled blade of a King Vision™ VL [Figure 1A–C]. After mask ventilation, a second laryngoscopy with the introduction of the standard blade of a King Vision™ VL through the mouth followed with gliding of the left DLT over the posterior surface of the standard non-channeled blade.\n\n(a) Arrow (1) shows how the proximal DLT curve remains directed to the right side. (b) Arrow (2) shows how the distal DLT curve follows the curve of the standard non-channeled blade (approximately 60–70°). (c) Shows the bronchial tip of the DLT adapted to the tip of the standard non-channeled blade.\n\nAfter satisfactory visualization, the left DLT was directed through the glottic opening into the trachea [Figure 2A–C]. The operator’s index finger prevented the perforation of the tracheal cuff of the DLT by the sharp upper teeth during passage through the mouth opening. The stylet was then removed and the DLT rotated counterclockwise 180° and advanced to the 27 cm mark at the incisors, while the glottis was visualized via the King Vision™ VL. The DLT position was verified fibreoptically.\n\nPhotograph showing a bronchial tip of a left 35 Fr double-lumen tube (DLT) passing towards (A) and through (B) the vocal cords, and (C) following removal of the stylet and 180° counterclockwise rotation of the DLT through the display unit of a King Vision™ videolaryngoscope.\n\nAnesthesia was maintained with sevoflurane (0.8–0.9 minimum alveolar concentration), TCI sufentanil with a Ce of 0.1 ng/mL and cisatracurium 5 mg. Transient severe hypotension (BP was 57–78/42–52 mm Hg that lasted for 25 min) was treated with reducing the sufentanil Ce to 0.05 ng/ml, and administering boluses of 6% hydroxyethyl starch 130/0.4 (Voluven® 6%, Hospira, Fresenius Kabi, Halden, Norway), as well as phenylephrine (300 µg) and ephedrine (10 mg).\n\nThe patient’s right lung was ventilated in pressure-controlled ventilation mode, with FiO2 set at 0.7, a delivered tidal volume (TV) of 360 mL, an inspiratory-to -expiratory [I: E] ratio of 1:2, PEEP of 5 cm H2O, and RR of 14–16/min. The peak airway pressure (Ppk) was limited to 35 cm H2O and a fresh gas flow (FGF) of 1.6 L/min was used. Neither continuous positive airway pressure (CPAP) nor high frequency positive pressure ventilation (HFPPV) was needed for the non-dependent lung5; SpO2 was maintained over 92% during 25 minutes of one lung ventilation (OLV). The operation proceeded uneventfully, with excellent lung isolation.\n\nAfter the surgery, the residual effects of neuromuscular blockade were reversed with neostigmine 2.5 mg and glycopyrrolate 0.6 mg. The patient was extubated and post-operative analgesia was accomplished with a continuous infusion of bupivacaine 0.125% through the paravertebral catheter. A post-operative follow-up (for the next six days after surgery) showed no evidence of hoarseness.\n\n\nDiscussion\n\nTwo main techniques can be used to achieve lung isolation in patients with a predicted difficult airway: [1] using a DLT or [2] using a bronchial blocker inserted through a SLT. There is no overall advantage of either over the other in the morbidly obese patient6.\n\nOur patient had predictable hypoxemia during OLV because of a severe restrictive pulmonary dysfunction and a low PaO2; despite this, significant hypoxemia was not noted during the relatively short period of OLV7. A DLT was chosen over a bronchial blocker so that the non-ventilated non-dependent lung could be oxygenated using HFPPV, although a bronchial blocker could have been used to provide CPAP to the non-ventilated non-dependent lung5. Additionally, a DLT allows effective bilateral suctioning. The difficulty in surgical access precluded the use of selective lower lobar collapse, which could have been helpful to correct the predicted intraoperative hypoxemia during OLV.\n\nWe used 6% hydroxyethyl starch 130/0.4 for treatment of hypotension, despite the concerns about the risk of acute kidney injury in critical ill patient, in part because a recent study showed a similar rate of acute kidney injury, coagulopathy and mortality with the use of NaCl 0.9% solution8.\n\nVideolaryngoscopy can sometimes facilitate DLT insertion compared with direct laryngoscopy9,10. Channeled VLs have many advantages over those with angulated blades, such as the GlideScope®. Channeled VLs have a passage to guide the SLT; thus, once an adequate view of the glottis has been obtained, the VL is kept steady and the SLT advanced into the glottis with the right hand. By contrast, the angulated blade design uses a different technique for placing the SLT: once an adequate view of the glottis is obtained, the operator holds the laryngoscope with the left hand and manipulates the SLT into the glottis with the right hand using the view on the screen is used as a guide11.\n\nChanneled videolaryngoscopes are more suitable in patients with a limited mouth opening compared to traditional videolaryngoscopes like the GlideScope®12. The King Vision™ VL accommodates a minimum mouth opening of 13 mm for the standard non-channeled blade and 18 mm for the channeled blade. Previous studies have demonstrated that the Airtraq® VL allows a better laryngeal visualization than the GlideScope®, making it potentially more effective for DLT placement13–15.\n\nHowever, the large outer diameter and more rigid design of DLTs make them relatively harder to insert it through classic channeled blades. This requires either the use of a specific videolaryngoscope design like the DLT version of the Airtraq®4, or the use of a tube exchanger over which a large DLT can be placed16. The DLT Airtraq® laryngoscope is available for the 35 Fr to 41 Fr DLTs. However, it has not gained widespread popularity because it requires a minimum mouth opening of 19 mm, provides only subtle enhancement of visualization, has a higher incidence of hoarseness over the Macintosh laryngoscopes4, and has a narrower field of view than King Vision™ VL (80° vs. 160°)17. Regardless, a superior field of view does not necessarily result in an improved view of the laryngeal inlet, or leads to easier insertion of the tracheal tube4.\n\nSuzuki et al. described the removal of the tube channel back plate of the Airway Scope® for intubation with a 39 Fr DLT in a patient with unpredicted difficult intubation and inadequate mouth opening18. Compared with the Airtraq® and the Pentax Airway Scope®, the standard non-channeled blade King Vision™ VL has the thinnest and shortest stature (26 mm vs. 28 mm and 49 mm and 13 mm vs. 18 mm and 131 mm, respectively) and the widest field of view (160° vs. 80° and 90°, respectively) that makes it superior for those with limited mouth opening17–19.\n\nAlthough the use of video laryngoscopy for placement of DLTs has been well described, the present report describes a novel approach to DLT intubation and offers another tool for patients who require lung isolation. The standard non-channeled blade of the King Vision™ VL could provide a new mean for insertion of DLTs in patients with a minimum mouth opening of 13 mm. This approach offers a 160° field of view, potentially facilitating the manipulation and rotation of the DLT upon visualization.\n\nHere we described the necessary maneuvers to insert a DLT using a standard non-channeled blade of King Vision™ VL. We recommend four steps: first, bend the DLT stylet so that the distal 21 cm of the DLT curve follows the curve of the standard non-channeled blade and the proximal curve of the DLT remains directed to the right side. Next, insert the DLT, exercising caution to avoid damage to the tracheal cuff by the upper teeth during its passage through the mouth opening. Then, after the bronchial cuff passes through the vocal cords, withdraw the stylet of the DLT. Finally, rotate the DLT 180° counterclockwise while advancing the DLT to the desired depth. In conclusion, the use of King Vision™ videolaryngoscope could offer an effective method of DLT placement for OLV.\n\n\nConsent\n\nThe patient provided informed written consent for the publication of this report.", "appendix": "Author contributions\n\n\n\nDrs Mohamed R El-Tahan and D. John Doyle contributed equally to this work as they were involved in designing the case presentation, and revised the manuscript critically for important intellectual content. Mohamed R El-Tahan reviewed the literature and wrote the manuscript. Mohamed R El-Tahan, Alaa M Khidr and Ahmed G Hassieb cared for the patient and revised the manuscript. Alaa M Khidr provided the patient’s pictures and is the author responsible for archiving the study files. Mohamed R El-Tahan submitted the final version of the manuscript. All authors approved the final version of the manuscript.\n\n\nCompeting interests\n\n\n\nAll authors declare that they have no conflicts of interest and received no financial support for the research, authorship, and/or publication of this article. Dr El Tahan received free airway device samples from Ambu in April 2014 for use in another study and he has no direct financial or other interest in Ambu (in the context of this and other studies).\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nHernandez AA, Wong DH: Using a Glidescope for intubation with a double lumen endotracheal tube. Can J Anaesth. 2005; 52(6): 658–659. PubMed Abstract \| Publisher Full Text\n\nChen A, Lai HY, Lin PC, et al.: GlideScope-assisted double-lumen endobronchial tube placement in a patient with unanticipated difficult airway. J Cardiothorac Vasc Anesth. 2008; 22(1): 170–172. PubMed Abstract \| Publisher Full Text\n\nRussell T, Slinger P, Roscoe A, et al.: A randomised controlled trial comparing the GlideScope(®) and the Macintosh laryngoscope fordouble-lumen endobronchial intubation. Anaesthesia. 2013; 68(12): 1253–1258. PubMed Abstract \| Publisher Full Text\n\nWasem S, Lazarus M, Hain J, et al.: Comparison of the Airtraq and the Macintosh laryngoscope for double-lumen tube intubation: a randomised clinical trial. Eur J Anaesthesiol. 2013; 30(4): 180–186. PubMed Abstract \| Publisher Full Text\n\nEl-Tahan MR, El Ghoneimy YF, Regal MA, et al.: Comparative study of the non-dependent continuous positive pressure ventilation and high-frequency positive-pressure ventilation during one-lung ventilation for video-assisted thoracoscopic surgery. Interact Cardiovasc Thorac Surg. 2011; 12(6): 899–902. PubMed Abstract \| Publisher Full Text\n\nCampos JH, Hallam EA, Ueda K: Lung isolation in the morbidly obese patient: a comparison of a left-sided double-lumen tracheal tube with the Arndt® wire-guided blocker. Br J Anaesth. 2012; 109(4): 630–635. PubMed Abstract \| Publisher Full Text\n\nLiu TJ, Shih MS, Lee WL, et al.: Hypoxemia during one-lung ventilation for robot-assisted coronary artery bypass graft surgery. Ann Thorac Surg. 2013; 96(1): 127–132. PubMed Abstract \| Publisher Full Text\n\nGuidet B, Martinet O, Boulain T, et al.: Assessment of hemodynamic efficacy and safety of 6% hydroxyethylstarch 130/0.4 vs. 0.9% NaCl fluid replacement in patients with severe sepsis: The CRYSTMAS study. Crit Care. 2012; 16(3): R94. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPurugganan RV, Jackson TA, Heir JS, et al.: Video laryngoscopy versus direct laryngoscopy for double lumen endotracheal tube intubation: a retrospective analysis. J Cardiothorac Vasc Anesth. 2012; 26(5): 845–848. PubMed Abstract \| Publisher Full Text\n\nYang M, Kim JA, Ahn HJ, et al.: Double-lumen tube tracheal intubation using a rigid video-stylet: a randomized controlled comparison with the Macintosh laryngoscope. Br J Anaesth. 2013; 111(6): 990–995. PubMed Abstract \| Publisher Full Text\n\nSavoldelli GL, Schiffer E, Abegg C, et al.: Learning curves of the Glidescope, the McGrath and the Airtraq laryngoscopes: a manikin study. Eur J Anaesthesiol. 2009; 26(7): 554–558. PubMed Abstract \| Publisher Full Text\n\nLiu L, Tanigawa K, Kusunoki S, et al.: Tracheal intubation of a difficult airway using Airway Scope, Airtraq, and Macintosh laryngoscope: a comparative manikin study of inexperienced personnel. Anesth Analg. 2010; 110(4): 1049–1055. PubMed Abstract \| Publisher Full Text\n\nPutz L, Dangelser G, Constant B, et al.: [Prospective trial comparing Airtraq and Glidescope techniques for intubation of obese patients]. Ann Fr Anesth Reanim. 2012; 31(5): 421–426. PubMed Abstract \| Publisher Full Text\n\nDarshane S, Ali M, Dhandapani S, et al.: Validation of a model of graded difficulty in Laerdal SimMan: functional comparisons between Macintosh, Truview EVO2, Glidescope Video Laryngoscope and Airtraq. Eur J Anaesthesiol. 2011; 28(3): 175–180. PubMed Abstract \| Publisher Full Text\n\nMcElwain J, Malik MA, Harte BH, et al.: Comparison of the C-MAC videolaryngoscope with the Macintosh, Glidescope, and Airtraq laryngoscopes in easy and difficult laryngoscopy scenarios in manikins. Anaesthesia. 2010; 65(5): 483–489. PubMed Abstract \| Publisher Full Text\n\nPoon KH, Liu EH: The Airway Scope for difficult double-lumen tube intubation. J Clin Anesth. 2008; 20(4): 319. PubMed Abstract \| Publisher Full Text\n\nWhite MC, Marsh CJ, Beringer RM, et al.: A randomised, controlled trial comparing the Airtraq™ optical laryngoscope with conventional laryngoscopy in infants and children. Anaesthesia. 2012; 67(3): 226–231. PubMed Abstract \| Publisher Full Text\n\nSuzuki A, Kunisawa T, Iwasaki H: Double lumen tube placement with the Pentax-Airway Scope. Can J Anaesth. 2007; 54(10): 853–854. PubMed Abstract \| Publisher Full Text\n\nSaracoglu KT, Eti Z, Gogus FY: Airtraq optical laryngoscope: advantages and disadvantages. Middle East J Anesthesiol. 2013; 22(2): 135–141. PubMed Abstract" }	[ { "id": "5164", "date": "30 Jun 2014", "name": "Maruyama Koichi", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for your case report demonstrating the effectiveness of non-channeled King Vision for DLT intubation in a morbidly obese patient with predicted difficult airway and severely restrictive pulmonary dysfunction. I have a few comments and questions:Was the patient pre-oxygenated enough? Oxygen at 3L/min via a nasal cannula could provide an approximate FiO2 of only 0.33. How long did it take from the cessation of mask ventilation until the resumption of the mechanical ventilation after intubation? In addition, could you tell me the lowest SpO2 during intubation? As you mentioned, the patient was easily expected to be exposed to the risk of severe hypoxia due to the morbid obesity and the comorbid pulmonary disease, as well as the predicted difficult airway. Furthermore, the time elapsed for intubation with the non-channeled King Vision would be longer than that with the conventional laryngoscopy or that with the channeled King Vision (Akihisa et al., 2014).The effectiveness of the non-channeled King Vision for DLT intubation was well described in this report. To clear the ethical issue, however, please provide the detailed information about the patient's safety.", "responses": [ { "c_id": "890", "date": "05 Jul 2014", "name": "Mohamed El Tahan", "role": "Reader Comment", "response": "We read with interest the important comments of Dr. Maruyama Koichi, Teikyo University, Japan. Here are our responses to his comments:Dr. Koichi: Was the patient pre-oxygenated enough? Oxygen at 3L/min via a nasal cannula could provide an approximate FiO2 of only 0.33. Authors: The initial delivered oxygen at 3 L/min via nasal cannulae was followed by routine preoxygenation with 100% oxygen in conjunction with 8% sevoflurane by mask ventilation. This lasted about 8 min. Dr. Koichi: How long did it take from the cessation of mask ventilation until the resumption of the mechanical ventilation after intubation? In addition, could you tell me the lowest SpO2 during intubation?Authors: The time taken from the cessation of mask ventilation until resumption of the mechanical ventilation after intubation lasted about 90s. The lowest SpO2 during intubation was 95%. Dr. Koichi: As you mentioned, the patient was easily expected to be exposed to the risk of severe hypoxia due to the morbid obesity and the comorbid pulmonary disease, as well as the predicted difficult airway. Furthermore, the time elapsed for intubation with the non-channeled King Vision would be longer than that with the conventional laryngoscopy or that with the channeled King Vision (Akihisa et al., 2014).Authors: First, as mentioned above, the apneic time lasted for only 90 seconds, with the lowest SpO2 being 95%. Second, we read with interest your cited study (Akihisa et al., 2014). In this study the authors reported longer intubation time with the use of the non-channelled King Vision laryngoscope compared with the conventional Macintosh or with the channelled King Vision video laryngoscopes (p < 0.001). However, the results of that particular study cannot readily be applied to the present case report because the operators were non-experienced nurses who had never previously performed tracheal intubation (rather than anesthesiologists with over 10 years of experience, as in the present report). In addition, the Akihisa study was done on manikins with simulated normal airways rather than patients with a difficult airway, and it tested the efficacy of the tested devices using a single lumen tube rather than the larger double lumen tube. Dr. Koichi: The effectiveness of the non-channeled King Vision for DLT intubation was well described in this report. To clear the ethical issue, however, please provide the detailed information about the patient's safety.Authors: First, as we mentioned, multidisciplinary discussions involving a cardiothoracic surgeon, a pulmonologist, anesthesiologists and the family of the patients took place, emphasizing the possibility of acute pulmonary compromise during tracheal intubation and surgery. Second, a stepwise plan was formulated as described. Third, standard monitoring, preoxygenation, and anesthetic technique were carried out. Fourth, the signed informed consent both for the perioperative management and for the publication of the patient’s data were obtained." } ] }, { "id": "5161", "date": "07 Jul 2014", "name": "David Healy", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this manuscript. The manuscript describes a single case report of the use of a video laryngoscope (King Vision non-channeled blade) to facilitate successful endobronchial intubation with a double lumen tube in an obese patients with respiratory comorbidity. The manuscript then discusses the characteristics of this particular videolayngoscope which the authors feel may lead to an improvement in success. This case report is well written and adequately describes this challenging clinical scenario and airway management. Technical issues:There is no documentation of attempt to perform bag-mask ventilation during the case. There is no discussion of why this was not attempted or of its importance in the overall airway management of this patient. My view is the ability to perform adequate bag mask ventilation may improve the patient’s oxygenation and safety during the intubation attempts; especially it they were less straightforward than that described in this manuscript. There was no attempt at direct laryngoscopy recorded. The actual difficulty of this patient’s airway remains unknown (e.g. she may have been easily ventilated with a bag mask and a grade I view at laryngoscopy with good positioning). She remains “at higher risk of difficulty” but the reality remains unknown. Content issues:I think the manuscript would benefit from a description of the other option of intubating a patient with a true difficult airway and significant pulmonary compromise: i.e. place a single lumen tube to improve oxygenation and ventilation, then exchange over an airway exchange catheter (Cook). I think the comment on fluid choice is unnecessary. Consider removing the phrase “Channeled VLs have many advantages over those with angulated blades, such as the GlideScope” - not true, and not referenced as so. Perhaps better described as “different advantages and limitations” Consider removing the phrase “Previous studies…..more effective for DLT” - the references do not back up the link between an improved view and “potential successful intubation”. This comment can falsely reinforce the idea that view (on VL) equates to success. I appreciate their relatively conservative summary statement at the articles conclusion - and the series of steps to achieve successful intubation.", "responses": [ { "c_id": "897", "date": "13 Jul 2014", "name": "Mohamed El Tahan", "role": "Reader Comment", "response": "We read with interest the important comments of Dr. David Healy, Department of Anesthesiology, University of Michigan, Ann Arbor, MI, USA. Here are our responses to his comments: Technical Issues:Dr. Healy: \"There is no documentation of attempt to perform bag-mask ventilation during the case. There is no discussion of why this was not attempted or of its importance in the overall airway management of this patient. My view is the ability to perform adequate bag mask ventilation may improve the patient’s oxygenation and safety during the intubation attempts; especially it they were less straightforward than that described in this manuscript.\"Authors: We mentioned that \"After positioning of the patient on the operating table in a head-up position, anesthesia was induced using a target-controlled infusion (TCI) of sufentanil with a target effect site concentration (Ce) of 0.1 ng/mL, in conjunction with 8% sevoflurane in oxygen delivered by mask ventilation.\". Thus mask ventilation was used during induction with pressure-support ventilation of 15 cm H2O and continued until the time laryngoscopy. We think that is unclear to the readers so we rewrote them again. We addressed that in the Case Description Section in the V2 of our report. Dr. Healy: \"There was no attempt at direct laryngoscopy recorded. The actual difficulty of this patient’s airway remains unknown (e.g. she may have been easily ventilated with a bag mask and a grade I view at laryngoscopy with good positioning). She remains “at higher risk of difficulty” but the reality remains unknown.\"Authors: We chose not to attempt direct laryngoscopy prior to the use of the King Vision™ VL. First, the patient had three predictors of an anticipated difficult airway, including a BMI of 41.7 kg/m2, a short thyromental distance and limited mouth opening. Second we were concerned about the possibility of hypoxemia if the duration of laryngoscopy and intubation was lengthened with a prior assessment of the airway using direct laryngoscopy. Note that the initial laryngoscopy using the King Vision™ VL revealed a Cormack-Lehane class II view, suggesting that the Cormack-Lehane class with direct laryngoscopy would be higher if this was used for an initial assessment. We thus felt that prior assessment of the airway using direct laryngoscopy would not be expected to change the adopted plan and would confer little benefit. Finally, note that the recent ASA Guidelines for Management of the Difficult Airway do not suggest a prior assessment using direct laryngoscopy. The guidelines also suggest the use of VL as a choice for tracheal intubation in the non-emergent pathway where ventilation is adequate.Content Issues:Dr. Healy: \"I think the manuscript would benefit from a description of the other option of intubating a patient with a true difficult airway and significant pulmonary compromise: i.e. place a single lumen tube to improve oxygenation and ventilation, then exchange over an airway exchange catheter (Cook).\"Authors: We have addressed this in the V2 in the Case Description as follows; Backup plans were revised involving the insertion of the left DLT over a placed Eschmann tracheal tube introducer (Smiths-Medical International Ltd, Hythe, Kent, UK), ), and using a King Vision™ VL, an Arndt’s endobronchial blocker placed through a SLT. Dr. Healy:\"I think the comment on fluid choice is unnecessary.”Authors: We deleted it from the Case Description in the V2. Dr. Healy: \"Consider removing the phrase “Channeled VLs have many advantages over those with angulated blades, such as the GlideScope” - not true, and not referenced as so. Perhaps better described as “different advantages and limitations”Authors': Unfortunately, it is “true” as Savoldelli et al (Reference no. 11 in the main text) have reported that time taken to position the endotracheal tube was shorter for the Airtraq (channelled VL) when compared with the Glidescope and McGrath (non-channelled VL). We amended the citation of that reference to be earlier. Dr. Healy: \"Consider removing the phrase “Previous studies…..more effective for DLT” - the references do not back up the link between an improved view and “potential successful intubation”. This comment can falsely reinforce the idea that view (on VL) equates to success.’Authors: The confusing sentence “, making it potentially more effective for DLT placement” has been removed. Dr. Healy: \"I appreciate their relatively conservative summary statement at the articles conclusion - and the series of steps to achieve successful intubation.Authors: Thank you so much. References:Enterlein G; Byhahn C. Practice guidelines for management of the difficult airway: update by the American Society of Anesthesiologists task force. Anaesthesist, 2013; 62(10): 832-5. Apfelbaum JL, Hagberg CA, Caplan RA, et al. Practice guidelines for management of the difficult airway: an updated report by the American Society of Anesthesiologists Task Force on Management of the Difficult Airway. Anesthesiology 2013;118(2): 251-70" } ] } ]	1	https://f1000research.com/articles/3-129
https://f1000research.com/articles/3-227/v1	29 Sep 14	{ "type": "Clinical Practice Article", "title": "Management of thrombocythemia", "authors": [ "Krisstina Gowin", "Ruben Mesa", "Krisstina Gowin" ], "abstract": "Essential thrombocythemia is a clonal myeloproliferative neoplasm characterized by an elevated platelet count, the potential for both microvascular and macrovascular sequelae, and a risk for transformation to myelofibrosis or acute myeloid leukemia. A systematic and detailed initial analysis is essential for accurate diagnosis of essential thrombocythemia, as many etiologies are reactive and benign. Once a diagnosis has been made, risk stratification and symptom assessment are vital to guide the subsequent therapy. Treatment may be required in high-risk disease, such as in cases of advanced age or prior thrombotic events. Systemic therapy is aimed at reducing the thrombotic risk and includes daily low dose aspirin and in some patients, cytoreductive therapy. Currently, the first line cytoreductive therapy includes hydroxyurea or pegylated interferon, with a phase III clinical trial underway comparing these two important agents. Anagrelide and clinical trials are reserved for refractory or intolerant patients. Looking to the future, new therapies including Janus kinase 2 (JAK2) and telomerase inhibitors are promising and may become valuable to the treatment armamentarium for those afflicted with essential thrombocythemia.", "keywords": [ "Thrombocythemia", "Janus kinase 2", "cytoreductive therapy" ], "content": "Introduction\n\nThrombocythemia, or elevation in platelet count (i.e. greater than 450 × 10 (9)/L), is a common observation for internists and hematologists alike. Causes may be secondary or “acquired” in contrast to primary thrombocythemia, meaning that the pathogenesis lies within the abnormal marrow itself. Essential thrombocythemia (ET), one of the myeloproliferative neoplasms (MPNs), is an aberration within the bone marrow and its microenvironment leading to clonal proliferation of the megakaryocytic lineage within the marrow and, ultimately, to peripheral blood thrombocythemia. Unlike secondary thrombocythemia, ET is associated with thrombotic and hemorrhagic complications and requires systemic medical therapy in high-risk patients. In this brief article, we discuss the diagnostic strategy of thrombocytosis with particular attention paid to essential thrombocythemia. Subsequently, the clinical manifestations of ET are examined and the assessment of disease burden is reviewed. The history of therapeutics for ET is reviewed with consideration to the current rationale for therapeutic decision-making.\n\n\nUncovering the etiology\n\nElucidating the etiology of thrombocythemia is of utmost importance prior to any therapeutic decision-making. Certainly, clonal bone marrow diseases such MPNs should be considered. However, such a diagnosis can only be considered after eliminating secondary contributions to the elevated platelet count. Many chronic and acute processes cause stimulation and up-regulation of bone marrow stem cells including infection, malignancy, iron deficiency, prior splenectomy, and recent trauma or surgery. (see Table 1). A careful examination of iron status, inflammatory markers, and age appropriate malignancy screen is imperative. History and physical exam, such as a history of gastrointestinal bleeding, rheumatologic disease or the presence of splenomegaly on exam can lend clues as to underlying etiologies. Once secondary causes are excluded, evaluation for an underlying clonal myeloproliferative disorder can commence.\n\n\nEstablishing a diagnosis\n\nMPNs such as ET, polycythemia vera (PV), and myelofibrosis (MF) are Philadelphia negative clonal disorders of the bone marrow1. When attempting to establish a diagnosis of MPN, mutational status can be quite helpful2. In 2005, a landmark discovery identified a gain of function mutation, JAKV617F, as being an essential mutational driver in many MPNs3–5. In ET, approximately 50% of patients will harbor the JAKV617F mutation. PCR based assays for the JAK2 mutation, from either peripheral blood or marrow, are commercially available. Since the JAKV617F discovery, other molecular breakthroughs have contributed not only to our knowledge of pathogenesis in MPNs but also how we diagnose them. In the majority of JAKV617F wild type patients, the CALR (calreticulin gene) mutation6 may be detected and now is a widely available assay. Additionally, MPL (myeloproliferative leukemia gene) mutations are detected in a small percentage (<5%) of those afflicted with ET7,8. Although mutation analysis is critical for the evaluation of a suspected MPN, it is not sufficient for diagnosis. A bone marrow biopsy must be obtained and possess features consistent with ET, such as megakaryocytic hyperplasia. Additionally, assessment of cytogenetics, baseline karyotype, reticulin fibrosis, and blast percentage should be performed. Mutational status for other myeloid diseases must be evaluated and negative including the BCR-ABL, i.e. “the Philadelphia chromosome”, and fluorescence in situ hybridization (FISH) for myelodysplastic syndrome (MDS) panel to exclude the diagnosis of CML and MDS, respectively (see Table 2)2.\n\n\nAssessing symptom burden\n\nThe presentation of MPN may be quite variable. A large proportion of those afflicted with ET are completely asymptomatic at presentation. Unfortunately, approximately 50% of patients with ET do possess some form of systemic manifestation of the disease and experience a substantial impact on their quality of life9. Common symptoms may include those from microvascular complications such as headache, dizziness, paresthesia, livedo reticularis, erythromelalgia, and visual changes. Others may present with the more dreaded macrovascular complications such as myocardial infarction, stroke, or pulmonary embolus. Additionally, constitutional symptoms may be prevalent with symptoms of fatigue, night sweats, and weight loss; particularly in those transitioning to a more myelofibrotic state. In 2007, a group of researchers set out to create and validate a symptom assessment tool specific to the MPN population10. Due to this effort, the MPN-Symptom Assessment Form (SAF)11 is now available and validated for use in this patient population and has proven to be an invaluable tool in the assessment and management of ET. Though use of the MPN-SAF a subset of ET patients were identified who possess a significant symptomatic burden, with fatigue and microvascular complications being the most prevalent10. For the treating clinician, the MPN-SAF can be utilized to assess baseline symptomatology and help guide initial therapeutic decision-making as well as gauge subsequent response to therapy.\n\n\nRisk assessment\n\nDeciding when to initiate therapy in ET may be complex and represents a unique challenge in the treatment of MPNs. A thrombotic risk assessment is necessary to evaluate whether initiation of cytoreduction is warranted12,13. The presence of high-risk features14, such as age greater than 60 years and a prior history of thrombosis, is predictive of future complications and generally prompts the clinician to employ cytoreduction. Additionally, concurrent cardiovascular risk factors15, JAK V617F mutational status and allelic burden16,17, and the presence of leukocytosis18 may increase the thrombotic risk potential and contribute to a clinician’s decision to initiate therapy. The presence of a heavy symptom burden may also provide more impetus to employ cytoreduction in afflicted patients who are otherwise in a low risk category. An international prognostic model for ET was developed in 2012 by Passamonti et al. and is helpful to ascertain risk and give valuable prognostic information to the treating physician (see Table 3)13. The treatment goal is improvement in disease related symptoms in addition to normalization of the platelet count to decrease thrombotic risk potential. Typically, the minimal effective dose is utilized to limit treatment-associated toxicity. In those with low-risk asymptomatic disease, simple observation is appropriate.\n\n\nInitial systemic therapy\n\nIn 2004, a European group investigated the use of aspirin for the prevention of thrombotic complications in PV and found that daily low dose aspirin can safely prevent thrombotic complications in those who have no contraindications to such treatment19. Since this landmark study, it is standard practice to administer daily low dose aspirin to all those with high-risk ET. In those with very high initial platelet counts, greater than 1,500/microL, an acquired Von Willebrand deficit may occur and increase risk for hemorrhagic complications. Because of this, some practitioners may elect to cytoreduce prior to aspirin initiation. Currently, first line cytoreductive therapy is a choice amongst three agents: hydroxyurea, anagrelide, and pegylated interferon. Fortunately, recent trials have clarified some therapeutic nuances of each choice. Hydroxyurea is a traditional treatment for preventing thrombosis in ET since Cortelazzo published on its efficacy in 199520. Later, anagrelide was approved for control of thrombocytosis based on single arm studies21. Subsequently, a conundrum was raised as to which agent was superior and preferential in first line therapy. In 2005, Harrison et al. sought to answer this with a randomized comparison of hydroxyurea to anagrelide22. In this study, hydroxyurea was found to be superior to anagrelide in terms of rate of arterial thrombosis, serious hemorrhage, and transformation to myelofibrosis, but was inferior in terms of rates of venous thrombosis. Consequently, hydroxyurea became standard first line therapy, with anagrelide being reserved for second line treatment. In 2008, pegylated interferon, a more tolerable form of interferon, was demonstrated to induce hematologic and molecular responses in ET23,24. As an added benefit, pegylated interferon has been shown to retard progression towards fibrosis in some studies25,26 however this remains controversial and is an area of ongoing investigation. Currently, it is still unknown whether hydroxyurea or pegylated interferon represents the best initial treatment strategy. The Myeloproliferative Disorders Research Consortium (MPD-RC) is conducting a phase III international study to evaluate the efficacy, safety, and tolerability of hydroxyurea versus pegylated interferon in frontline therapy for ET/PV. (clinicaltrials.gov: NCTO1259817). Additionally, it is important to mention that interferon therapy is safe in pregnancy, unlike hydroxyurea and anagrelide and thus, pegylated interferon is the preferred agent in this patient population or those who wish to become pregnant.\n\n\nSecond line therapy\n\nIn those who are intolerant or resistant to initial therapy a therapeutic switch is indicated and is largely guided by first line choices. A common practice is to progress through the first line cytoreductive agents, with no data directing the sequence of therapies. Aspirin is continued throughout if not contraindicated. The duration of therapy is typically lifelong, with the goal of treatment being hemorrhagic and thrombotic risk reduction, as well as retardation of disease progression. For those who are intolerant to or progressed on all approved agents, clinical trials should be considered. Novel therapeutics, particularly JAK inhibitors, offer a valuable addition to the treatment armamentarium and are available via clinical trial for ET. Moreover, other drug classes such as telomerase inhibitors are promising for the future treatment of ET. Often, in those with very proliferative disease (i.e. platelet count >2000 × 10(9)/L), an effective combination therapeutic approach is used. Hydroxyurea and anagrelide, for example, can be used concurrently for optimal cytoreduction and greater tolerability, as the dosage of each is lower in combination than with single agent therapy alone.\n\n\nMonitoring for progression\n\nA minority of patients progress to myelofibrosis or acute myeloid leukemia (AML)27. Practitioners should pay careful attention to the patient’s symptom burden, peripheral blood counts, and cytogenetic analysis for clues indicating progression. The development of increased constitutional symptoms such as progressive splenomegaly, fever, weight loss, early satiety, and bone pain in conjunction with a trend towards either new cytopenia or increased rate of proliferative disease increases clinical suspicion of a post ET-myelofibrosis. Conversely, those with new blasts on peripheral smear and/or marrow and new cytogenetic complexity should be evaluated for MPN blast phase or AML28.\n\n\nConclusion\n\nIn evaluating cases of thrombocythemia, it is essential to exclude both reactive processes and other chronic myeloid disorders prior to making the diagnosis of essential thrombocythemia. Mutational analysis is helpful in making the diagnosis and the well-informed clinician can consider JAKV617F and if wild type subsequent MPL, and CALR assessment in new patient evaluations. In ET patients with high-risk disease, aspirin plus either hydroxyurea versus pegylated interferon is the standard first line therapy. Anagrelide is appropriate as an adjunct to therapy or for second line usage. Clinical trial enrollment is imperative to answer outstanding questions regarding safety, tolerability, and efficacy of alternative therapies29 including JAK2 and telomerase inhibitors, both of which have demonstrated promising early results in the treatment of ET.", "appendix": "Author contributions\n\n\n\nKrisstina Gowin: Authored manuscript, literature review\n\nRuben Mesa: Edits\n\nBoth authors agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nTefferi A, Thiele J, Vardiman JW: The 2008 World Health Organization classification system for myeloproliferative neoplasms: order out of chaos. Cancer. 2009; 115(17): 3842–7. PubMed Abstract \| Publisher Full Text\n\nVardiman JW, Thiele J, Arber DA, et al.: The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood. 2009; 114(5): 937–51. PubMed Abstract \| Publisher Full Text\n\nKralovics R, Passamonti F, Buser AS, et al.: A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med. 2005; 352(17): 1779–90. PubMed Abstract \| Publisher Full Text\n\nLevine RL, Wadleigh M, Cools J, et al.: Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell. 2005; 7(4): 387–97. PubMed Abstract \| Publisher Full Text\n\nJames C, Ugo V, Le Couedic JP, et al.: A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature. 2005; 434(7037): 1144–8. 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PubMed Abstract \| Publisher Full Text\n\nEmanuel RM, Dueck AC, Geyer HL, et al.: Myeloproliferative neoplasm (MPN) symptom assessment form total symptom score: prospective international assessment of an abbreviated symptom burden scoring system among patients with MPNs. J Clin Oncol. 2012; 30(33): 4098–103. PubMed Abstract \| Publisher Full Text\n\nScherber R, Dueck AC, Johansson P, et al.: The Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF): international prospective validation and reliability trial in 402 patients. Blood. 2011; 118(2): 401–8. PubMed Abstract \| Publisher Full Text\n\nBarbui T, Barosi G, Birgegard G, et al.: Philadelphia-negative classical myeloproliferative neoplasms: critical concepts and management recommendations from European LeukemiaNet. J Clin Oncol. 2011; 29(6): 761–70. PubMed Abstract \| Publisher Full Text\n\nPassamonti F, Thiele J, Girodon F, et al.: A prognostic model to predict survival in 867 World Health Organization-defined essential thrombocythemia at diagnosis: a study by the International Working Group on Myelofibrosis Research and Treatment. Blood. 2012; 120(6): 1197–201. PubMed Abstract \| Publisher Full Text\n\nBarbui T, Finazzi G, Carobbio A, et al.: Development and validation of an International Prognostic Score of thrombosis in World Health Organization-essential thrombocythemia (IPSET-thrombosis). Blood. 2012; 120(26): 5128–33; quiz 5252. PubMed Abstract \| Publisher Full Text\n\nTefferi A: Polycythemia vera and essential thrombocythemia: 2013 update on diagnosis, risk-stratification, and management. Am J Hematol. 2013; 88(6): 507–16. PubMed Abstract \| Publisher Full Text\n\nAntonioli E, Guglielmelli P, Pancrazzi A, et al.: Clinical implications of the JAK2 V617F mutation in essential thrombocythemia. Leukemia. 2005; 19(10): 1847–9. PubMed Abstract \| Publisher Full Text\n\nGangat N, Wassie E, Lasho T, et al.: Mutations and thrombosis in essential thrombocythemia: prognostic interaction with age and thrombosis history. Eur J Haematol. 2014. PubMed Abstract \| Publisher Full Text\n\nBarbui T, Finazzi G, Falanga A: Myeloproliferative neoplasms and thrombosis. Blood. 2013; 122(13): 2176–84. PubMed Abstract \| Publisher Full Text\n\nLandolfi R, Marchioli R, Kutti J, et al.: Efficacy and safety of low-dose aspirin in polycythemia vera. N Engl J Med. 2004; 350(2): 114–24. PubMed Abstract \| Publisher Full Text\n\nCortelazzo S, Finazzi G, Ruggeri M, et al.: Hydroxyurea for patients with essential thrombocythemia and a high risk of thrombosis. N Engl J Med. 1995; 332(17): 1132–6. PubMed Abstract \| Publisher Full Text\n\nSteurer M, Gastl G, Jedrzejczak WW, et al.: Anagrelide for thrombocytosis in myeloproliferative disorders: a prospective study to assess efficacy and adverse event profile. Cancer. 2004; 101(10): 2239–46. PubMed Abstract \| Publisher Full Text\n\nHarrison CN, Campbell PJ, Buck G, et al.: Hydroxyurea compared with anagrelide in high-risk essential thrombocythemia. N Engl J Med. 2005; 353(1): 33–45. PubMed Abstract \| Publisher Full Text\n\nKiladjian JJ, Cassinat B, Chevret S, et al.: Pegylated interferon-alfa-2a induces complete hematologic and molecular responses with low toxicity in polycythemia vera. Blood. 2008; 112(8): 3065–72. PubMed Abstract \| Publisher Full Text\n\nQuintas-Cardama A, Kantarjian H, Manshouri T, et al.: Pegylated interferon alfa-2a yields high rates of hematologic and molecular response in patients with advanced essential thrombocythemia and polycythemia vera. J Clin Oncol. 2009; 27(32): 5418–24. PubMed Abstract \| Publisher Full Text\n\nSilver RT, Vandris K, Goldman JJ: Recombinant interferon-α may retard progression of early primary myelofibrosis: a preliminary report. Blood. 2011; 117(24): 6669–72. PubMed Abstract \| Publisher Full Text\n\nTefferi A, Elliot MA, Yoon SY, et al.: Clinical and bone marrow effects of interferon alfa therapy in myelofibrosis with myeloid metaplasia. Blood. 2001; 97(6): 1896. PubMed Abstract \| Publisher Full Text\n\nMesa RA, Silverstein MN, Jacobsen SJ, et al.: Population-based incidence and survival figures in essential thrombocythemia and agnogenic myeloid metaplasia: an Olmsted County Study, 1976–1995. Am J Hematol. 1999; 61(1): 10–5. PubMed Abstract \| Publisher Full Text\n\nMesa RA, Verstovsek S, Cervantes F, et al.: Primary myelofibrosis (PMF), post polycythemia vera myelofibrosis (post-PV MF), post essential thrombocythemia myelofibrosis (post-ET MF), blast phase PMF (PMF-BP): Consensus on terminology by the international working group for myelofibrosis research and treatment (IWG-MRT). Leuk Res. 2007; 31(6): 737–40. PubMed Abstract \| Publisher Full Text\n\nFiskus W, Ganguly S, Kambhampati S, et al.: Role of additional novel therapies in myeloproliferative neoplasms. Hematol Oncol Clin North Am. 2012; 26(5): 959–80. PubMed Abstract \| Publisher Full Text" }	[ { "id": "6242", "date": "03 Oct 2014", "name": "Laura C. Michaelis", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well-written review that addresses some of the key considerations in the diagnosis and treatment of ET. The authors are right to emphasize the importance of a bone marrow biopsy in diagnosis, as it is important to exclude early PMF, which can have a different disease trajectory. In addition, practitioners should also be aware of the disproportionately high rates of splanchnic vein or mesenteric thromboses, which can be a presenting symptom in these patients and should prompt clinicians to search for MPNs.", "responses": [] }, { "id": "6243", "date": "13 Oct 2014", "name": "Ann Mullally", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nNice summary of clinical management of ET.", "responses": [] } ]	1	https://f1000research.com/articles/3-227
https://f1000research.com/articles/3-161/v1	11 Jul 14	{ "type": "Opinion Article", "title": "Estrogen as Jekyll and Hyde: regulation of cell death", "authors": [ "Wen Zhou", "Xiaoxia Zhu", "Xiaoxia Zhu" ], "abstract": "Estrogen has profound effects on growth, differentiation and function in male and female reproductive systems, and it is important for bone density, brain function and cholesterol mobilization. Despite beneficial estrogen functions, sustained estrogenic exposure increases the risk and/or the progression of various cancers, including those of the breast, endometrium and ovary. This opinion article touches upon the dual role estrogen played in cancer and asks whether the use of estrogen in combination with other targeted therapy would be possible, considering the newly identified crosstalk pathway which can switch the effects of estrogen.", "keywords": [ "17β-estradiol", "breast cancer", "apoptosis", "IKKα", "BAY11-7082" ], "content": "Introduction\n\nOur research projects currently focus on understanding of the interplay between the different signaling cascades and estrogen receptor (ER) dependent transcription activation in breast cancer. For many years, those of us working in the field were used to looking at estrogen as a mitogen through its genomic function mediated by the ER-dependent transcription program. Now we realize that estrogen through ER, in addition to regulate gene expression, crosstalks with many non-genomic signaling pathways involved in cell growth, differentiation and apoptosis. These newly identified interplays may give a different flavor to long conceived mitogenic role of estrogen. In this opinion article, we generally reviewed the changing attitude toward estrogen in clinical use, with a focus on a new discovery that estrogen, in combination with IKKα, can induce breast cancer cell apoptosis effectively. We also discuss the possibility of estrogen and IKKα inhibitor dual-therapy strategy in cancer treatment.\n\n\nHistorical perspective and changing attitude to estrogen in clinical use\n\nSir George Thomas Beatson (1896) used oophorectomy to reduce the estrogen level in premenopausal women in order to prevent breast cancer occurrence, and he was the first to reveal the relationship between estrogen levels and breast cancer1. Half a century later, Haddow et al. (1944) first used a high-dose synthetic estrogen (stillbestrol) to induce tumor regression in hormone-dependent breast cancer in postmenopausal women2. Huggins et al. (1952) pioneered adrenalectomy to reduce estrogen level for treating mammary cancer3. Huggins’ work is internationally recognized by the prestigious Nobel Prize (1966) for the contribution to the development of endocrine therapy in hormone-regulated cancer. Jensen E (1958) characterized the first receptor for estrogen - estrogen receptor alpha (ERα). Soon after these discoveries, extensive mechanistic studies have gained large information about estrogen’s physiological functions and carcinogenic roles. The Women's Health Initiative (WHI) research program (1991) initiated a 15-year study enrolled 161,808 generally healthy women aged 50–79 to evaluate the beneficial effects of postmenopausal hormone replacement therapy (HRT) on heart diseases, bone fractures, and cancers4. Due to the increased incidence of breast cancer, stroke, and cardiovascular complications in women treated with estrogen alone or with a combination of estrogen and progesterone, the study was terminated prematurely in 2002. Though extensively studied, the definite understanding of the mechanism of estrogen action always challenges our mind.\n\n\nThe challenge: paradoxical role of estrogen in cell death\n\nEstrogen regulates the proliferation and development of tissues expressing estrogen receptors and ERα is mainly expressed in breast epithelium, ovary and endometrium. Thus, estrogen is mitogenic for cultured ER positive breast cancer lines. The mitogenic effects of estrogen at the G1-to-S transition are mediated by the key effectors of estrogen action, c-Myc, cyclin D1 and E2F-15–7. c-Myc expression occurs within 15 min of estrogen stimulation, among the earliest responses to estrogen. Estrogen also rapidly induces cyclin D1 expression. In the G1 phase, estrogen drives E2F-1 expression. Estrogen-triggered all these coherent genetic changes to guarantee the cell cycle progression. Nongenomically, Estrogen binding to the ER stimulates rapid activation of Src and signaling pathways MAPK and PI3K/Akt pathways that affect cell survival8,9. Based on these understandings of estrogen action, ER protein is assayed in newly diagnosed breast cancers because it is a clinically useful prognostic factor and predicts responsiveness to ER blocking drugs such as tamoxifen.\n\nParadoxically, estrogen induces apoptosis under certain circumstances. As mentioned above, high-dose estrogen was used to induce tumor regression of hormone-dependent breast cancer in postmenopausal women before the introduction of tamoxifen2. This regimen is of clinical interest, given that long-term treatment of breast cancer with anti-estrogen drug tamoxifen often leads to drug resistance and that sustained tamoxifen exposure may sensitize breast cancer cells to high-dose or even low-dose estrogen therapy10. The field of the mysterious dual effects of estrogen on apoptosis have not much progressed until recently.\n\n\nRecent research breakthrough\n\nRecently, Perillo’s Group from Second University of Naples identified a key player, IKKα in the switch of estrogen action in apoptosis11. They found that ER agonist 17β-estradiol (E2) and IKKα kinase specific inhibitor BAY11-7082 (BAY) in combination can induce apoptosis in an ERα-positive breast cancer cell line. Dual-therapy now receives more and more attention.\n\nIn the journal Cell Death & Differentiation, Perillo et al. recently reported that the inhibition of IKKα by BAY switched the effect of estrogens on breast cancer cells from anti- to pro-apoptotic, which leads the exploration of therapeutic benefits of estrogen into a new era11. IKKα is the kinase responsible for histone H3 Ser 10 phosphorylation (H3pS10)12. H3pS10 is known to inhibit H3 Lys 9 dimethylation (H3K9me2) in a space repulsion model13. Thus, inhibiting H3pS10 by targeting IKKα facilitates estrogen-triggered ER-dependent recruitment of histone methyltransferase Suv39H1. Histone demethylase LSD1 demethylating the Suv39H1 target sites H3K9me2 was increased concomitantly. LSD1-mediated demethylation process is known to produce reactive oxygen species (ROS) and cause ROS-mediated DNA damaging effects14. The net results after IKKα knowndown is causing more DNA damages to cancer cells through estrogen triggered ER-dependent Suv39H1 and LSD1 binding to ER target gene promoter (Figure 1).\n\nUpper panel: chromatin landscape and factors present at the pS2 (as known as TFF1) promoter in the presence of IKKα. The pS2 promoter is enriched with nucleosomes (blue and white cylinders) that dwell in positions proximal to the transcription start site (+1 position) and at ER binding sites. Only low levels of histone H3 lysine 9 dimethylation (H3K9me2) exist due to the space repulsion of histone methyltransferases binding to H3K9 from IKKα residing at the neighboring H3S10 site. RNA polymerase holoenzyme (Pol II) (yellow oval) is present at the proximal promoter region near the transcription start site (TSS, shown by black vertical line) of the pS2 gene. Lower panel: chromatin landscape and factors present at pS2 promoter following IKKα knockdown or IKKα inhibitor BAY11-7082 treatment. Once levels of IKKα have decreased, ER recruits the histone methyltransferase Suv39H1 or demethylase LSD1 proteins to bind within the pS2 promoter. Once the LSD1 is activated and demethylates its target H3K9me2, it generates reactive oxygen species (ROS) to cause DNA damage effects including base oxidation and nicks results from DNA damage itself and related DNA repair. In sum, the inhibition of IKKα results in the reversion of estrogen triggered anti-apoptotic effects to pro-apoptotic effects.\n\nIn short, Perillo's group identifies a novel crosstalk between IKKα and estrogen signaling and shows that inhibition of IKKα-mediated histone phosphorylation switch ER-mediated anti-apoptotic effects to ER-dependent ROS-mediated breast cell death, which implicates potential dual-therapy of ER agonist (E2) together with IKKα inhibitor (BAY) in a variety of hormone-regulated cancers.\n\n\nFuture perspectives\n\nIn the last few years, there have been significant shifts in the attitudes towards the use of estrogen in clinic. Estrogen exhibits a broad range of functions that regulates cell proliferation and homeostasis in many tissues. Despite beneficial estrogen functions, sustained estrogenic exposure increases the risk and/or the progression of various cancers, including those of the breast, endometrium and ovary15. The International Agency for Research on Cancer (IARC) has listed estrogen as known human carcinogen16. Now, the success of the combination of E2 and BAY will certainly become an “accelerator” to the alternative use of estrogen in treating cancers and we expect to see more positive pre-clinical and/or clinical results in the near future.", "appendix": "Author contributions\n\n\n\nConceived and designed the Figure: WZ XZ. Wrote the paper: WZ XZ\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was partially supported by US Department of Defense pre-doctoral grant W81XWH-11-1-0097 (W.Z.).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors thank laboratory members for helpful discussions.\n\n\nReferences\n\nBeatson GT: On treatment of inoperable cases of carcinoma of the mamma: suggestions for a new method of treatment with illustrative cases. Lancet. 1896; 148(3802): 104–7. Publisher Full Text\n\nHaddow A, Watkinson JM, Paterson E, et al.: Influence of Synthetic Oestrogens on Advanced Malignant Disease. Br Med J. 1944; 2(4368): 393–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuggins C, Bergenstal DM: Effect of Bilateral Adrenalectomy on Certain Human Tumors. Proc Natl Acad Sci U S A. 1952; 38(1): 73–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrentice RL, Anderson GL: The women’s health initiative: lessons learned. Annu Rev Public Health. 2008; 29: 131–50. PubMed Abstract \| Publisher Full Text\n\nMusgrove EA, Caldon CE, Barraclough J, et al.: Cyclin D as a therapeutic target in cancer. Nat Rev Cancer. 2011; 11(8): 558–72. PubMed Abstract \| Publisher Full Text\n\nCaldon CE, Musgrove EA: Distinct and redundant functions of cyclin E1 and cyclin E2 in development and cancer. Cell Div. 2010; 5: 2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou W, Srinivasan S, Nawaz Z, et al.: ERα, SKP2 and E2F-1 form a feed forward loop driving late ERα targets and G1 cell cycle progression. Oncogene. 2014; 33(18): 2341–53. PubMed Abstract \| Publisher Full Text\n\nSun J, Zhou W, Kaliappan K, et al.: ERα phosphorylation at Y537 by Src triggers E6-AP-ERα binding, ERα ubiquitylation, promoter occupancy, and target gene expression. Mol Endocrinol. 2012; 26(9): 1567–77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKato S, Endoh H, Masuhiro Y, et al.: Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science. 1995; 270(5241): 1491–4. PubMed Abstract \| Publisher Full Text\n\nAriazi EA, Cunliffe HE, Lewis-Wambi JS, et al.: Estrogen induces apoptosis in estrogen deprivation-resistant breast cancer through stress responses as identified by global gene expression across time. Proc Natl Acad Sci U S A. 2011; 108(47): 18879–86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPerillo B, Di Santi A, Cernera G, et al.: Phosphorylation of H3 serine 10 by IKKα governs cyclical production of ROS in estrogen-induced transcription and ensures DNA wholeness. Cell Death Differ. 2014. PubMed Abstract \| Publisher Full Text\n\nPark KJ, Krishnan V, O’Malley BW, et al.: Formation of an IKKalpha-dependent transcription complex is required for estrogen receptor-mediated gene activation. Mol Cell. 2005; 18(1): 71–82. PubMed Abstract \| Publisher Full Text\n\nRea S, Eisenhaber F, O–Carroll D, et al.: Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature. 2000; 406(6796): 593–599. PubMed Abstract \| Publisher Full Text\n\nPerillo B, Ombra MN, Bertoni A, et al.: DNA oxidation as triggered by H3K9me2 demethylation drives estrogen-induced gene expression. Science. 2008; 319(5860): 202–206. PubMed Abstract \| Publisher Full Text\n\nZhou W, Slingerland JM: Links between oestrogen receptor activation and proteolysis: relevance to hormone-regulated cancer therapy. Nat Rev Cancer. 2014; 14(1): 26–38. PubMed Abstract \| Publisher Full Text\n\nIARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Combined estrogen-progestogen contraceptives and combined estrogen-progestogen menopausal therapy. IARC Monogr Eval Carcinog Risks Hum. 2007; 91: 1–528. PubMed Abstract" }	[ { "id": "5459", "date": "11 Aug 2014", "name": "Steven L Young", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this opinion manuscript, Zhou and Zhu highlight recent research that suggests estrogen can act on opposing signaling pathways to either promote proliferation or cell death in breast cancer cells and, further, that the signaling can be biased toward cell death by inhibition of IKKa action. The manuscript is thought provoking and timely and the argument is logical and generally well-constructed. The title is eye-catching and appropriate. The figure is attractive and highlights the key point of the manuscript. If the areas of concern (below) are addressed, the conclusions will be justified by the argument presented.I have four areas of concern:The manuscript is marred by frequent grammatical and English language usage errors as well as occasional typographic errors. While readable, it is sometimes difficult to determine the precise meaning of the authors’ statements. In the introduction, the authors state that estrogen, in combination with IKKa, can induce breast cancer apoptosis. However, it is IKKa inhibition that is used, suggesting that it is the absence of IKKa effect that is important - this should be clarified as currently the penultimate sentence of the introduction paragraph appears to be contradicted by the sentence following. The authors propose that IKKa inhibition has great promise for clinical use, but fail to remark on factors that would be important for feasibility of such therapy (see below): If IKKa were inhibited systemically, would apoptosis be largely limited to the cancer cells? What about other estrogen target tissues? Are there known toxicities of BAY or other IKKa inhibitors that would need to be considered?\n\nAre the concentrations of estradiol and IKKa inhibitor necessary to induce cell death achievable in humans? Are the concentrations of estradiol required for induction of cell death in the normal pre-menopausal range, or are these pharmacological levels: important implications for treatment of women with functioning ovaries and for the thrombogenic side effects of estrogen therapy, which are dose-related.4. The abstract does not effectively summarize the manuscript.", "responses": [ { "c_id": "1007", "date": "25 Sep 2014", "name": "Wen Zhou", "role": "Reader Comment", "response": "\"In this opinion manuscript, Zhou and Zhu highlight recent research that suggests estrogen can act on opposing signaling pathways to either promote proliferation or cell death in breast cancer cells and, further, that the signaling can be biased toward cell death by inhibition of IKKa action. The manuscript is thought provoking and timely and the argument is logical and generally well-constructed. The title is eye-catching and appropriate. The figure is attractive and highlights the key point of the manuscript. If the areas of concern (below) are addressed, the conclusions will be justified by the argument presented.\"We thank Dr. Young for his supportive and encouraging comments.Major concerns:\"The manuscript is marred by frequent grammatical and English language usage errors as well as occasional typographic errors. While readable, it is sometimes difficult to determine the precise meaning of the authors’ statements.\"We made the corrections. \"[T]he authors state that estrogen, in combination with IKKa, can induce breast cancer apoptosis. However, it is IKKa inhibition that is used, suggesting that it is the absence of IKKa effect that is important - this should be clarified as currently the penultimate sentence of the introduction paragraph appears to be contradicted by the sentence following.\"We agree with Dr. Young and we have made appropriate changes. \"The abstract does not effectively summarize the manuscript.\"We fully agree with Dr. Young. We re-wrote the abstract part to give more accurate summary of the content and include several recent related literature. We made also changes to the discussion part in accordance to these new findings.Additional concerns:\"If IKKa were inhibited systemically, would apoptosis be largely limited to the cancer cells? What about other estrogen target tissues?\"Though the toxicity of IKKα inhibition is unknown, IKKα-mediated activity has been implicated in cerntain cancers. In addition, IKKα has been reported to enhance RelA/p65 activity, which protein was implicated to drive oncogenic transformations. Like selective ER modulators (SERM) therapy, this therapy may have adverse effect on other estrogen target tissues. This merits further investigation. \"Are there known toxicities of BAY or other IKKa inhibitors that would need to be considered?\"The toxicities of BAY11-7082 (BAY) is unknown. Most IKK inhibitors are highly selective for β isoform, and there are few IKKα-specific inhibitors. Further investigation on its toxicity is highly needed. \"Are the concentrations of estradiol and IKKa inhibitor necessary to induce cell death achievable in humans?\"The concentration of estradiol used in Perillo’s study is 10 nM, and IKKα inhibitor BAY used is 20 µM. The same concentration of estradiol is achievable in humans. The human pharmacokinetic data for BAY is not available, though BAY is also shown to be effective when used in 10 µM or even 1 µM concentrations (Che et al., 2014; Zhao et al., 2014; Jung et al., 2014). \"Are the concentrations of estradiol required for induction of cell death in the normal pre-menopausal range, or are these pharmacological levels: important implications for treatment of women with functioning ovaries and for the thrombogenic side effects of estrogen therapy, which are dose-related.\"The concentrations of estradiol required when used together with BAY for the induction of cell death is 10 nM, which is within the normal premenopausal range. We agree with Dr. Young and also another reviewer Dr. Philippa Sauders that it is important to take into account the different hormonal effect on pre- or postmenopausal women, and the dose-related thrombogenic side effect of estrogen." } ] }, { "id": "6196", "date": "22 Sep 2014", "name": "Philippa Saunders", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nNew ideas about the way oestrogens might affect the growth, differentiation and functional development of cancers, including those of the breast, are always welcome. The authors' Opinion Article questions whether giving oestrogens (rather than the more common anti-oestrogens) may merit exploration as an alternative treatment for breast cancers when provided in combination with agents that alter inflammatory signalling pathways. A limitation of the commentary is that it has focused on data from studies using breast cancer cell lines such as the MCF-7 cells cited in the studies by the Perillo group 1-3. A limitation of all studies using tissue-culture adapted cell lines such as MCF-7 is that they do not reflect the genomic heterogeneity we now appreciate exists in different breast tumours 4, only a proportion of which are considered classically activated by oestrogens because they are ER alpha positive. In spite of these reservations the authors do raise some interesting points. Firstly, they highlight the potential for inflammatory mediators such as those that activate the NFκB signalling pathway as targets for therapies in breast cancer treatment. One example provided is the data from the Perillo group, reporting that inhibition of IKKα kinase specific inhibitor BAY11-7082 might be beneficial in switching the effects of oestrogens from anti- to pro-apoptotic. These studies are in agreement with those from a number of other authors claiming that combination therapies are likely to be beneficial in treatment of this malignancy. Notably, there is an interesting paper by Biswas et al. published in 2004 5 which examines how NFκB activation in breast cancer specimens might have a role in cell proliferation and apoptosis. Notably, in the Biswas paper, they report results suggesting that activated NFκB is predominantly detected in ER negative rather than ER positive breast tumours. This paper, as well as the recent genomic proflling of breast cancers from 2000 patients 4 highlights the importance of considering the both the immediate microenvironment of the tumour as well as the adjacent non-tumour tissue. Notably, in considering how oestrogen therapy might be utilised, it is important to take into account the hormonal status of the woman (pre or post menopausal) as well as the compelling data that has demonstrated intra-tissue oestrogen biosynthesis in fat and other cells close to the malignant epithelial cells in situ 6. A paper that has just appeared in the Journal of Clinical Endocrinology and Metabolism by Savolainen-Peltonen and colleagues 7 is particularly relevant. In this paper, the authors compared the metabolic pathways producing active oestradiol in breast subcutaneous adipose tissue of postmenopausal women with and without cancer, and these showed there were differences depending on whether the adipose tissue was proximal or distal to a tumour. If the tumour is already bathed in high concentrations of oestrogen either from the periphery or local biosynthesis then addition of more ligand is unlikely to be effective. The local inflammatory environment of the tumour is an important factor in tumour progression and it is influenced by activation of NFκB activity leading to production of inflammatory mediators such as TNFα, IL-1β and prostaglandin E2 from adipocyteassociated macrophages and COX-2 inhibitors have been cited as effective in reducing recurrence of cancer 8. Local increases in inflammatory mediators in turn stimulate adipocyte aromatase expression/activity and hence oestradiol production 9 that may be particularly relevant to obese women 9,10. Thus, when considering suppression of NFκB activity in combination therapy there may be many context-dependent impacts on the tumour microenvironment to consider. The role of NFκB activation as a regulator of inflammatory signalling is particularly important to consider in the context of endocrine resistant breast cancer which cannot be addressed using MCF-7 cells. A new paper in Molecular Cellular Endocrinology by Litchfield et al. 11 notes that regulation of endocrine-resistant breast cancer cells may also be mediated by other transcription factors such as COUPTFII. So the keys questions to be addressed in considering the value of the future prospective advanced within this Opinion piece are the following:Are studies in isolated cell lines such as the one described really relevant when it comes to translating new therapies into practice? Is inhibition of inflammatory mediators such as that using the BAY compound really most effective in cells that are not actually estrogen responsive?This merits further investigation. Finally, how can we move forward with the field? In our opinion, an excellent way to advance these studies would be to conduct further investigations using methods which better reflect the microenvironment of an intact tumour in situ, this should include studies on the interplay between malignant epithelial cells, fat and vascular and inflammatory cells as this likely to influence response to therapy and it cannot be under-estimated that the individual responses to oestrogens by these different cell types may vary considerably. Examples of such studies currently include 3 being explored in the context of breast cancer, including organoids, tissue slices or xenografts. These approaches are more likely than studies in MCF-7 cells to inform choice of new combination therapies for testing in clinical trials; oestrogen combined with NFκB inhibition should be tested in these systems 12-14.", "responses": [ { "c_id": "1008", "date": "25 Sep 2014", "name": "Wen Zhou", "role": "Reader Comment", "response": "The observations made by Dr. Saunders were very pertinent and allowed us to alter the manuscript in several key points. Below we address each one of the reviewer comments. We hope to have provided all the information needed regarding the work submitted and that the manuscript is now suitable for publishing and archiving. Alterations were signalized in the text.Responses:\"A limitation of the commentary is that it has focused on data from studies using breast cancer cell lines such as the MCF-7 cells cited in the studies by the Perillo group. A limitation of all studies using tissue-culture adapted cell lines such as MCF-7 is that they do not reflect the genomic heterogeneity we now appreciate exists in different breast tumours, only a proportion of which are considered classically activated by oestrogens because they are ER alpha positive … Are studies in isolated cell lines such as the one described really relevant when it comes to translating new therapies into practice?\"We thank Dr. Saunders for her comment. Perillo used an experimental approach to evaluate the molecular interaction of both ER and other signaling pathways in MCF-7 cells, as described in many journal publications (Garcia et al., 1992; Planas-Silva et al., 1999; Sun et al., 2012). In these works an evaluation of the biological effect of the molecular interaction was assessed as Perillo group performed in their work. Thus, we believe Perillo’s cell line based studies are meaningful and relevant to find novel targets in hormone responsive cancers. Still, we have altered the manuscript, avoiding the reference to other proportion of breast tumors which are not so responsive to hormone therapy. Although it has been previously reported that ER positive cancers make up to two thirds of newly diagnosed breast cancer cases, we agree with Dr. Saunders that it is important to appreciate the highly existed heterogeneity under the name of breast cancer, to take into account the hormone status of women, and to better understanding the tumor-stromal microenvironment. \"Notably, there is an interesting paper by Biswas et al. published in 2004 which examines how NFκB activation in breast cancer specimens might have a role in cell proliferation and apoptosis. Notably, in the Biswas paper, they report results suggesting that activated NFκB is predominantly detected in ER negative rather than ER positive breast tumours… Is inhibition of inflammatory mediators such as that using the BAY compound really most effective in cells that are not actually estrogen responsive? \"We agree with Dr. Saunders that activated NFκB is predominantly detected in ER negative rather than ER positive breast tumors. Perillo’s study we referred in our opinion article also support this point, as “BAY (IKKα inhibitor) only” induced more apoptosis in MDA-MB-231 (ER-negative) cells compared with MCF-7 in their Figure S1C. However, the point to make here, as shown in original Perillo paper, is that estradiol combined with BAY has been more potent in inducing apoptosis in ER positive cells compared with each agent alone in either cell lines. Perillo’s study revealed the previously hidden pro-apoptotic property of estrogen in physiological concentration range. On this stand, it inspired us to write this opinion article, and we think it was an interesting observation and worth attracting more attention as well as the warm discussions we have thus far achieved. \"Finally, how can we move forward with the field? In our opinion, an excellent way to advance these studies would be to conduct further investigations using methods which better reflect the microenvironment of an intact tumour in situ, this should include studies on the interplay between malignant epithelial cells, fat and vascular and inflammatory cells as this likely to influence response to therapy and it cannot be under-estimated that the individual responses to oestrogens by these different cell types may vary considerably. Examples of such studies currently include 3 being explored in the context of breast cancer, including organoids, tissue slices or xenografts. These approaches are more likely than studies in MCF-7 cells to inform choice of new combination therapies for testing in clinical trials; oestrogen combined with NFκB inhibition should be tested in these systems.\"We thank Dr. Saunders for her comments and have changed part of our content according to her suggestion." } ] } ]	1	https://f1000research.com/articles/3-161
https://f1000research.com/articles/3-226/v1	23 Sep 14	{ "type": "Research Article", "title": "Preoperative low-dose ketamine has no preemptive analgesic effect in opioid-naïve patients undergoing colon surgery when nitrous oxide is used - a randomized study", "authors": [ "Beatriz Nistal-Nuño", "Enrique Freire-Vila", "Francisco Castro-Seoane", "Manuel Camba-Rodriguez", "Enrique Freire-Vila", "Francisco Castro-Seoane", "Manuel Camba-Rodriguez" ], "abstract": "Background: The analgesic properties of ketamine are associated with its non-competitive antagonism of the N-methyl-D-aspartate receptor; these receptors exhibit an excitatory function on pain transmission and this binding seems to inhibit or reverse the central sensitization of pain. In the literature, the value of this anesthetic for preemptive analgesia in the control of postoperative pain is uncertain. The objective of this study was to ascertain whether preoperative low-dose ketamine reduces postoperative pain and morphine consumption in adults undergoing colon surgery.Methods: In a double-blind, randomized trial, 48 patients were studied. Patients in the ketamine group received 0.5 mg/kg intravenous ketamine before surgical incision, while the control group received normal saline. The postoperative analgesia was achieved with a continuous infusion of morphine at 0.015 mg∙kgˉ¹∙hˉ¹ with the possibility of 0.02 mg/kg bolus every 10 min. Pain was assessed using the Visual Analog Scale (VAS), morphine consumption, and hemodynamic parameters at 0, 1, 2, 4, 8, 12, 16, and 24 hours postoperatively. We quantified times to rescue analgesic (Paracetamol), adverse effects and patient satisfaction.Results: No significant differences were observed in VAS scores between groups (P>0.05), except at 4 hours postoperatively (P=0.040). There were no differences in cumulative consumption of morphine at any time point (P>0.05). We found no significant differences in incremental postoperative doses of morphine consumption in bolus, except at 12 h (P =0.013) and 24 h (P =0.002). The time to first required rescue analgesia was 70 ± 15.491 min in the ketamine group and 44 ± 19.494 min in the control (P>0.05). There were no differences in hemodynamic parameters or patient satisfaction (P>0.05).Conclusions: Preoperative low-dose-ketamine did not show a preemptive analgesic effect or efficacy as an adjuvant for decreasing opioid requirements for postoperative pain in patients receiving intravenous analgesia with morphine after colon surgery.", "keywords": [ "colon surgery", "ketamine", "patient-controlled-analgesia", "preemptive analgesia" ], "content": "Introduction\n\nIn spite of the techniques we have at our disposal and the elementary nature of incisional pain, optimal pain management remains a challenge1. Because the severity of early postoperative pain relates to residual pain after some types of surgery, perioperative pain management can considerably influence the long-term quality of life in patients2,3.\n\nWoolf, in 1983, first introduced the theory of preemptive analgesia to attenuate postoperative pain4, confirming the presence of a central factor of post-injury pain hypersensitivity in experimental research. After this, experimental studies showed that various anti-nociceptive methods applied before injuries were more effective in reducing post-injury central sensitization in contrast to administration after injury5.\n\nAfter activation of C-fibers by noxious stimuli, sensory neurons become more sensitive to peripheral inputs, a process called central sensitization6,7. ‘Wind up’8, another mechanism activating spinal sensory neurons, is seen after reiterated stimulation of C-fibers. These sensitizations produce c-fos expression in sensory neurons9, and are related to the activation of N-methyl-D-aspartic acid (NMDA)7,9 and neurokinin receptors10,11. These genes produce long-lasting changes in the pain-processing system, resulting in hyperexcitation. According to Wall, protection of sensory neurons against central sensitization may provide relief from pain after surgery12. Based on this assumption, preemptive analgesia has been recommended as an effective aid to control postsurgical pain4,13,14. NMDA antagonists have been demonstrated to block the induction of central sensitization and revoke the hypersensitivity once it is established7,15.\n\nKetamine is an old drug that is increasingly being considered for the treatment of acute and chronic pain. Its pharmacology and mechanism of action as an NMDA receptor antagonist are adequately known, but in clinical practice it presents irregular results16. Since ketamine is an NMDA-receptor antagonist, it is supposed to avoid or revoke central sensitization, and thus to attenuate postoperative pain17.\n\nThis antihyperalgesic action can be achieved by smaller doses than those required for anesthesia. Small-dose ketamine has been specified as not more than 1 mg/kg when given as an iv bolus, and not higher than 20 µg∙kg-1∙min-1 when given as a constant infusion18,19.\n\nLow-doses preemptive ketamine administered iv seem to reduce postoperative pain and/or analgesic consumption15,20,21. According to one study19, a single dose of ketamine 1 mg/kg, when administered in conjunction with local anesthetics, opioids or other anesthetics, provides good postoperative pain control17.\n\nRegardless of the overwhelming effectiveness of preemptive ketamine in animal experiments22–24, clinical reports are mixed25–29; some authors have described positive effects30 while others have not31.\n\nWhile early reviews of clinical findings were mostly contradictory26,32, there is still conviction among researchers and clinicians in the effectiveness of preemptive analgesia5.\n\nTo our knowledge, no prior controlled study has determined the effectiveness of preoperative low-dose iv ketamine as contrasted with placebo in adults after open colon surgery. Thus, this clinical trial was designed to examine the postoperative analgesic effectiveness and opioid-sparing effect of single low-dose iv ketamine in contrast with placebo administered preoperatively.\n\n\nMethods\n\nAfter receiving authorization from the Institutional Ethics Committee (Protocol code MK334037) and according to Helsinki, Tokyo, and Venezia statements, 48 patients undergoing general anesthesia for open colon surgery at the C. Hospitalario Arquitecto Marcide - Profesor Novoa Santos, were studied. This was a randomized controlled clinical trial, ClinicalTrials.gov identifier: NCT02241278.\n\nStudy candidates were identified from the surgery schedule and contacted for consent 1–7 days before surgery. All patients gave written, informed consent, after explanation about the objectives, methods and potential risks of the study. Procedures included open colon resections, right hemicolectomy and left hemicolectomy.\n\nInclusion criteria were age between 18 and 75 years, normal Body Mass Index (18.5–24.9), ASA class I, II or III, elective surgery, surgery time between 60–150 min, understanding of the Visual Analog Scale (VAS), lack of allergies or intolerance to anesthetics and absence of psychiatric illness. Exclusion criteria included cognitive deterioration, inability to use the Patient-Controlled-Analgesia (PCA) device, history of chronic pain syndromes or chronic use of analgesics, sedatives, opioids or steroids, liver or hematologic disease, history of drug or alcohol abuse and intolerance to ketamine or Paracetamol.\n\nPatients were instructed preoperatively on the use of the VAS for pain assessment and the PCA device. The VAS represents a scale with the lowest value as 0 (no pain) and the highest value as 10 (worst imaginable pain).\n\nRandomization was based on computer-produced random-block codes maintained in successively numbered envelopes and organized in a double-blinded manner. Pharmacy-prepared 50 mL solutions containing either ketamine (0.5 mg/kg) or placebo were given to anesthesiologists. The anesthesiologists and patients were not aware of the treatment groups. The investigator, unaware of the treatment groups and not implicated in patient’s intraoperative care, performed postoperative assessments.\n\nAll subjects were premedicated with metoclopramide 10 mg and ranitidine 300 mg v.o. the night before and at 07.00 h on the day of surgery, and with diazepam 5–10 mg v.o. the night before surgery. In the operating room, the anesthesiologist administered 0.5 mg/kg of ketamine chlorhydrate in 0.9% saline iv to patients in the ketamine group and 50 mL of 0.9% saline to the control group 30 minutes before surgical incision. Besides routine monitoring, the patients were monitored with spectral entropy through an Entropy Module (M-Entropy TM; Datex-Ohmeda, Helsinki, Finland) and muscle relaxation (M-NMT module).\n\nAfter premedication with atropine 0.01 mg/kg if necessary, general anesthesia was induced with propofol 1–2 mg/kg (or thiopental 6 mg/kg), remifentanyl at 0.5 µg∙kg-1∙min-1 iv (0.25 µg∙kg-1∙min-1 in patients over 65 years old), muscle relaxation with succinilcoline 1 mg/kg or cisatracurium 0.15 mg/kg. Anesthesia was maintained with nitrous oxide 50% and sevoflurane 0.5–1% in 50% oxygen, remifentanyl in continuous infusion at 0.5–1 µg∙kg-1∙min-1, and neuromuscular blockage with cisatracurium in bolus of 0.06 mg/kg on demand. Anesthesia was adjusted to keep arterial blood pressure and heart rate within 20% of preinduction levels. 30 min before surgical closure, 0.10 mg/kg of morphine was administered iv; a continuous infusion of morphine (PCA) was initiated at 0.015 mg∙kg-1∙h-1 and planned to deliver a bolus of 0.02 mg/kg of morphine on demand, with a lockout interval of 10 min. The infusion of remifentanyl was stopped at the end of surgery. Decurarization if necessary was achieved with atropine 0.01 mg/kg and neostigmine 0.03 mg/kg. The use of opioid reversal agents, different analgesics to the ones studied and other treatments that could interfere with the pain evaluation was not permitted. Patients were extubated in the operating room and moved to the Post-Anesthesia Care Unit (PACU).\n\nPain severity was evaluated at time 0 (at entrance in the PACU), and at 1, 2, 4, 8, 12, 16, and 24 hours postoperatively. Pain was graded using the VAS. If VAS >5, a rescue dose of Paracetamol 1 gr iv was given as rescue analgesia. The cumulative amounts of morphine administered through the PCA as a basal infusion and the incremental supplemental bolus required by the patient were documented at these same time points. Hemodynamic parameters such as Blood Pressure (BP) systolic, BP diastolic, heart rate and respiratory rate were measured at these same time points. The time interval for the first demand of analgesia and the number of times a rescue dose was injected in the first 24 hours were recorded. Global patient satisfaction (0–3), regarding pain control, was measured 24 hours after the operation. All adverse effects and their characteristics were recorded.\n\nPrior to the study, we calculated the sample size needed for justifying the assumption that postoperative pain (VAS) would be less in the ketamine group than in the control (primary outcome measure). A mean difference in VAS scores of 2.05 (assuming a target of 20.5% reduction in VAS scores) between groups in the first 24 hours postoperatively was defined as clinically relevant. This criterion was based on the results of a previous pilot study at our institution using the same surgical population and the same outcomes. The required sample size to reveal clinically relevant reductions was estimated to be 24 patients per category, giving a statistical power of 0.80 and a type I error protection of 0.05.\n\nWe performed a descriptive analysis, presenting the numerical variables as mean ± standard deviation and the categorical variables as integer values and percentages.\n\nCategorical variables were contrasted between groups with the Chi-square test. Numerical variables were compared between groups, after checking the assumption of normal distribution with the Kolmogorov-Smirnov test, with the Student’s t-test test or the Mann-Whitney U-test accordingly.\n\nVariables in the different time points were compared with the Friedman test for related groups. The level of significance was established at P<0.05. Data were examined utilizing SPSS statistical software (v.19.0).\n\n\nResults\n\nA total of 48 patients were recruited during 8 months and completed the study. All patients were discharged and no patients presented any severe postoperative complications.\n\nNo significant differences were observed between the two groups in demographics such as ASA group (P=1.000), sex (P=0.745) or age (P=0.177). However, they were different in weight (P=0.015) [Table 1]. The two groups did not deviate in terms of duration of the surgical procedure (P=0.701), intraoperative doses of remifentanyl (P=0.861) or intraoperative doses of morphine (P=0.572). [Table 2].\n\nValues are mean ± SD except gender distribution (frequency) and ASA physical status (median value).\n\nan = 24\n\nValues are mean ± SD.\n\nan = 24\n\nThere were no statistically significant differences in VAS scores between the groups, except at 4 hours of arrival to the PACU, when the scores in the ketamine group were higher than in the control group (P=0.040). We could see a significant effect of time in pain scores for each group separately (P<0.001) [Figure 1]. On arrival at the PACU, pain intensity was higher in the control group, becoming maximal at 1 hour but with higher scores in the ketamine group at this time. We could observe a progressive decrease in pain scores afterwards.\n\n(Mean ± SD). There were no statistically significant differences between the groups, except at 4 hours of arrival at the PACU (P=0.040)*. We could see a significant effect of time in pain scores for each group separately (P<0.001).\n\nNo significant differences were assessed between the two groups in cumulative consumption of morphine at any time point during the first postoperative 24 hours (P>0.05 at all time points).The effect of time on morphine consumption through PCA in the entire postoperative period was not statistically significant (P>0.05). (Figure 2).\n\n(Mean ± SD). There were no significant differences between groups at any time point (P>0.05). The effect of time on total morphine consumption in the postoperative period was not statistically significant (P>0.05).\n\nThe amount of incremental postoperative doses of morphine consumption in bolus from the PCA was comparable in the two groups. We found no statistically significant differences among groups, except at 12 h (P=0.013) and 24 h (P=0.002). It seems the need of additional boluses of morphine over the basal infusion rate of the PCA was slightly higher in the ketamine group at all time points, except immediately after arrival at the PACU (Figure 3). The total amount of bolus supplements of morphine needed throughout the 24 h was higher in the ketamine group than in the control group (P=0.02). The time to first solicited rescue analgesia was 70 ± 15.491 min in the ketamine group (6 patients) and 44 ± 19.494 min in the control group (5 patients) (P=0.052).\n\n(Mean ± SD). There were no statistically significant differences among groups at any time point, except at 12 h (P=0.013) and 24 h (P=0.002).\n\nNo discordances in patient satisfaction were detected between the groups (P>0.05). The majority of patients rated their pain control as excellent across the 24 h after the operation.\n\nSecondary effects encountered in the ketamine group were nausea (5 patients), urinary retention (1 patient), vomiting (1 patient), incoercible vomiting (1 patient). In the control group they were nausea (3 patients) and urinary retention (2 patients). The differences among groups were not significant (P>0.05). No patient encountered any side effects interpreted as severe (Table 3).\n\nAdverse effects are expressed as number of patients.\n\nan = 24\n\nWhen evaluating the hemodynamic parameters as an indirect measure of pain, we found the following results. The BP systolic at all time points during the postoperative 24 h was very similar between both groups (P>0.05 at all time points). We could appreciate a slight increase of BP systolic on arrival at the PACU, with a progressive decrease over the 24 h until final stabilization. The BP diastolic was comparable between both groups, with no major statistical deviations, except at 0 h (P=0.026), 8 h (P=0.02) and 24 h (P=0.02), being higher in the ketamine group. These differences did not appear to be clinically significant. The respiratory rate showed no differences between both groups, except at 0 h, being higher in the placebo group (P=0.027), but this difference was not clinically significant. There were no significant differences among groups in heart rate (P>0.05 at all time points).\n\n\nDiscussion\n\nDemonstration for a clinically significant preemptive analgesic effect of low-dose ketamine is questionable33. Studies have shown a preemptive effect15,21,28,34 and others have not18,26,27,31,35,36. Some authors found a 40% decrease in PCA morphine consumption21 and a decrement in hyperalgesia 48 hours37 and 7 days38 after surgery. Barbieri et al.39 recorded lower VAS results until 24 hours after elective laparoscopy for ovarian cysts in patients given 1 mg/kg im ketamine before surgery. Fu et al.15 contrasted the analgesic effect of a presurgical loading dose (0.5 mg/kg), followed by a constant infusion (10 µg∙kg-1∙min-1) with a single postsurgical dose (0.5 mg/kg). They found a significant decrease in PCA morphine consumption 48 h after surgery in the preemptive group26.\n\nWe can deduce from our results that no significant intergroup distinction was encountered in the pain scores. Neither a morphine-sparing effect nor a lower mean supplemental dose of morphine through the PCA was demonstrated at any point in time in the ketamine group.\n\nDespite these results, we observed good analgesia in the immediate postoperative period; as reflected in the pain scores, which were low in both groups and within the maximum limits of VAS 3–4.5; these scores are usually assumed as adequate. As clinically evaluated, there was no activation of the sympathetic nervous system induced by pain in the postoperative period, evidenced by the lack of significant rises in blood pressure, heart rate or respiratory rate. Also, the incidence of adverse effects was low.\n\nStill, we expected that if ketamine had a preemptive analgesic effect, this would have become apparent in the immediate postoperative stage, with significantly lower consumption of morphine and lower pain scores in that group18. However, we cannot unequivocally conclude that ketamine has no preemptive effect from the above information.\n\nA possible explanation is the anesthetic procedure. As debated by Katz40 and Dahl41, examinations on preemptive substances should attempt to clarify whether these substances have a postoperative analgesic effect when clinically pertinent anesthesia, including perioperative opioids, have also been delivered. In all patients, anesthesia was induced and maintained with remifentanil. This may have hidden the preemptive analgesia of ketamine26.\n\nAnimal and human investigations propose that the use of adjuvant drugs as part of general anesthesia can act on the central sensitizing effects of surgical stimuli, making it more complex to discern a preemptive effect27,42,43. Since even short phases of C-fiber stimulation from surgical injury can lead to sensitization of the central nervous system44, it seems that the constant intraoperative administration of opioids would be superior to reiterated boluses. In our study, the perioperative administration of opioids (remifentanil and morphine) could have blocked, at the presynaptic opioid receptors at the terminals of the C fibers, the release of afferent transmitters involved in pain transmission. Thus, the administration of an NMDA receptor antagonist may have been redundant18.\n\nMoreover, anesthesia was maintained with nitrous oxide in both groups, which has been shown to diminish nociception-induced spinal sensitization in rats26,42,45 and to show a preemptive analgesic effect13,42. Experimental evidence exists in rats that nitrous oxide does block spinal sensitization42, perhaps by the same mechanism as opioids46. However, Goto et al.42 demonstrated that halothane and isoflurane47 moderately antagonize this effect equally. Nevertheless, some studies using oxygen/nitrous oxide have exhibited a preemptive analgesic effect13,48,49.\n\nAnother potential problem was the small dose of drug administered, which might have caused a deficient afferent antinociceptive blockade in the preemptive group. This small dosage has a brief length of action, and central sensitization may have been generated when the pharmacological action of ketamine ended26.\n\nSensitization is a persistent phenomenon, conditional to the amplitude and length of the nociceptive stimulus. Our study centered on major surgery, where deep noxious stimuli continues during surgery and may even extend postoperatively. The best method to avoid sensitization may be to intercept any pain from the time of incision until final lesion recovering26. Nonetheless, the psychomimetic effects of ketamine limit the clinical value of large-dose ketamine27,50.\n\nAs suggested by the study of Subramaniam et al.51, ketamine acts primarily on opened ionic channels to prevent neuroplasticity52. When the drug is given prior to surgery, the channels are not in an open phase, because no noxious stimulus is present. Therefore, it is conceivable that ketamine, because of its brief length of action, must be given as a continuous infusion to inhibit the intraoperative noxious stimuli and the ‘wind up’ occurrence15,51.\n\nReza et al.53 described in their work that postoperative morphine need was not diminished when 0.5 mg/kg ketamine was given preemptively. Ngan Kee et al. illustrated that the postoperative analgesic demand was diminished when 1.0 mg/kg was given in their study54. In spite of this, in other studies 0.5 mg/kg of ketamine was useful for alleviating postoperative pain after abdominal surgery5,15, and in others the need for analgesia after cesarean section was diminished with administration of a low dosage of 0.15 mg/kg55,56. In another article the morphine demand was similar in three categories of cesarean section subjects given 0.25, 0.5, or 1.0 mg/kg of ketamine57; hence, it is plausible that the preemptive analgesic action of ketamine might not be dose conditional58.\n\nThe choice of surgical procedure may also help to explain our results. Low intensity noxious stimuli during surgery may not incite sufficient central sensitization to create a clear difference between the study groups33. Laskowski et al.59 concluded from their study that the efficacy of ketamine was superior in upper abdominal operations, thoracotomy, or if the VAS score was ≥ 7, in contrast to lower abdominal surgery or if the VAS score was < 4. After colon surgery, pain intensity is moderate and may not create adequate highly noxious stimulus to ascertain any clear differences between groups26.\n\nIn conclusion, this study failed to exhibit a preemptive analgesic effect of 0.5 mg/kg iv preoperative ketamine, showing no significant advantage on postoperative pain and analgesic consumption. Thus, further comparative and controlled studies of the effects of higher doses in larger study sizes are required before definitive recommendations can be presented.\n\n\nClinical trial registration statement\n\nPatient enrollment for this clinical trial took place during the years 2001 and 2002. The study was not registered prospectively prior to patient enrollment because at the time the trial began enrollment of subjects (years 2001–2002) it was not mandatory the registration of clinical trials on account of the Spanish regulations. The trial was registered on 09/11/2014.\n\n\nData availability\n\nF1000Research: Dataset 1. Data on the effect of preoperative low-dose ketamine in opioid-naïve patients undergoing colon surgery when nitrous oxide is used, 10.5256/f1000research.5258.d3561660", "appendix": "Author contributions\n\n\n\nBeatriz Nistal-Nuño analyzed the data, wrote the manuscript and participated in the design of the study. Statistical data analysis, interpretation of results and subsequent discussion, writing all sections of the manuscript including its translation and editing (abstract, introduction, methods, results, discussion, references, figures and tables). She is the author responsible for archiving the study files.\n\nEnrique Freire Vila monitored the study and collected the data.\n\nFrancisco Castro Seoane designed and conducted the study, and collected the data.\n\nManuel Camba Rodriguez initiated the study.\n\nAll authors have seen the original study data, reviewed the analysis of the data, and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nSupplementary materials\n\nCONSORT checklist regarding the clinical trial described in this article. Click here to access this file.\n\n\nReferences\n\nBrennan TJ: Frontiers in translational research: the etiology of incisional and postoperative pain. Anesthesiology. 2002; 97(3): 535–7. PubMed Abstract \| Publisher Full Text\n\nPerkins FM, Kehlet H: Chronic pain as an outcome of surgery. A review of predictive factors. 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PubMed Abstract \| Publisher Full Text\n\nMurray WB, Yankelowitz SM, le Roux M, et al.: Prevention of post-tonsillectomy pain with analgesic doses of ketamine. S Afr Med J. 1987; 72(12): 839–42. PubMed Abstract\n\nRoytblat L, Korotkoruchko A, Katz J, et al.: Postoperative pain: the effect of low-dose ketamine in addition to general anesthesia. Anesth Analg. 1993; 77(6): 1161–5. PubMed Abstract \| Publisher Full Text\n\nWoolf CJ: Evidence for a central component of post-injury pain hypersensitivity. Nature. 1983; 306(5944): 686–8. PubMed Abstract \| Publisher Full Text\n\nBennett GJ: Update on the neurophysiology of pain transmission and modulation: focus on the NMDA-receptor. J Pain Symptom Manage. 2000; 19(1 Suppl): S2–6. PubMed Abstract \| Publisher Full Text\n\nEide PK: Wind-up and the NMDA receptor complex from a clinical perspective. Eur J Pain. 2000; 4(1): 5–15. PubMed Abstract \| Publisher Full Text\n\nHeinke W, Grimm D: Preemptive effects caused by co-analgesia with ketamine in gynecological laparotomies? Anaesthesiol Reanim. 1999; 24(3): 60–4. PubMed Abstract\n\nAdam F, Libier M, Oszustowicz T, et al.: Preoperative small-dose ketamine has no preemptive analgesic effect in patients undergoing total mastectomy. Anesth Analg. 1999; 89(2): 444–7. PubMed Abstract \| Publisher Full Text\n\nDahl V, Ernoe PE, Steen T, et al.: Does ketamine have preemptive effects in women undergoing abdominal hysterectomy procedures? Anesth Analg. 2000; 90(6): 1419–22. PubMed Abstract \| Publisher Full Text\n\nMenigaux C, Fletcher D, Dupont X, et al.: The benefits of intraoperative small-dose ketamine on postoperative pain after anterior cruciate ligament repair. Anesth Analg. 2000; 90(1): 129–35. PubMed Abstract \| Publisher Full Text\n\nKwok RF, Lim J, Chan MT, et al.: Preoperative ketamine improves postoperative analgesia after gynecologic laparoscopic surgery. Anesth Analg. 2004; 98(4): 1044–9, table of contents. PubMed Abstract \| Publisher Full Text\n\nPapaziogas B, Argiriadou H, Papagiannopoulou P, et al.: Preincisional intravenous low-dose ketamine and local infiltration with ropivacaine reduces postoperative pain after laparoscopic cholecystectomy. Surg Endosc. 2001; 15(9): 1030–3. PubMed Abstract \| Publisher Full Text\n\nMathisen LC, Aasbø V, Raeder J: Lack of pre-emptive analgesic effect of (R)-ketamine in laparoscopic cholecystectomy. Acta Anaesthesiol Scand. 1999; 43(2): 220–4. PubMed Abstract \| Publisher Full Text\n\nLebrun T, Van Elstraete AC, Sandefo I, et al.: Lack of a pre-emptive effect of low-dose ketamine on postoperative pain following oral surgery. Can J Anaesth. 2006; 53(2): 146–52. PubMed Abstract \| Publisher Full Text\n\nKissin I: Preemptive analgesia. Why its effect is not always obvious. Anesthesiology. 1996; 84(5): 1015–9. PubMed Abstract \| Publisher Full Text\n\nSuzuki M, Tsueda K, Lansing PS, et al.: Small-dose ketamine enhances morphine-induced analgesia after outpatient surgery. Anesth Analg. 1999; 89(1): 98–103. PubMed Abstract \| Publisher Full Text\n\nVan Elstraete AC, Lebrun T, Sandefo I, et al.: Ketamine does not decrease postoperative pain after remifentanil-based anaesthesia for tonsillectomy in adults. Acta Anaesthesiol Scand. 2004; 48(6): 756–60. PubMed Abstract \| Publisher Full Text\n\nVan Elstraete AC, Lebrun T, Sandefo I, et al.: Are preemptive analgesic effects of ketamine linked to inadequate perioperative analgesia? Anesth Analg. 2004; 99(5): 1576. PubMed Abstract \| Publisher Full Text\n\nTverskoy M, Oz Y, Isakson A, et al.: Preemptive effect of fentanyl and ketamine on postoperative pain and wound hyperalgesia. Anesth Analg. 1994; 78(2): 205–9. PubMed Abstract\n\nStubhaug A, Breivik H, Eide PK, et al.: Mapping of punctuate hyperalgesia around a surgical incision demonstrates that ketamine is a powerful suppressor of central sensitization to pain following surgery. Acta Anaesthesiol Scand. 1997; 41(9): 1124–32. PubMed Abstract \| Publisher Full Text\n\nBarbieri M, Colnaghi E, Tommasino C, et al.: Efficacy of the NMDA antagonist ketamine in preemptive analgesia. In: Jensen TS, Turner JA, Wiesenfeld-Hallin Z, eds. Proceedings of the 8th world congress on pain. Seattle: IASP Press, 1997: 343–9.\n\nKatz J: Pre-emptive analgesia: evidence, current status and future directions. Eur J Anaesthesiol Suppl. 1995; 10: 8–13. PubMed Abstract\n\nDahl JB: The status of pre-emptive analgesia. Curr Opin Anaesthesiol. 1995; 8(4): 323–30.Publisher Full Text\n\nGoto T, Marota JJ, Crosby G: Nitrous oxide induces preemptive analgesia in the rat that is antagonized by halothane. Anesthesiology. 1994; 80(2): 409–16. PubMed Abstract \| Publisher Full Text\n\nWilson RJ, Leith S, Jackson IJ, et al.: Pre-emptive analgesia from intravenous administration of opioids. No effect with alfentanil. Anaesthesia. 1994; 49(7): 591–3. PubMed Abstract \| Publisher Full Text\n\nDickenson AH: Spinal cord pharmacology of pain. Br J Anaesth. 1995; 75(2): 193–200. PubMed Abstract \| Publisher Full Text\n\nO’Connor TC, Abram SE: Inhibition of nociception-induced spinal sensitization by anesthetic agents. Anesthesiology. 1995; 82(1): 259–66. PubMed Abstract \| Publisher Full Text\n\nGuo TZ, Poree L, Golden W, et al.: Antinociceptive response to nitrous oxide is mediated by supraspinal opiate and spinal alpha 2 adrenergic receptors in the rat. Anesthesiology. 1996; 85(4): 846–52. PubMed Abstract \| Publisher Full Text\n\nAbram SE, Yaksh TL: Morphine, but not inhalation anesthesia, blocks post-injury facilitation. The role of preemptive suppression of afferent transmission. Anesthesiology. 1993; 78(4): 713–21. PubMed Abstract \| Publisher Full Text\n\nKatz J, Clairoux M, Kavanagh BP, et al.: Pre-emptive lumbar epidural anaesthesia reduces postoperative pain and patient-controlled morphine consumption after lower abdominal surgery. Pain. 1994; 59(3): 395–403. PubMed Abstract \| Publisher Full Text\n\nFletcher D, Zetlaoui P, Monin S, et al.: Influence of timing on the analgesic effect of intravenous ketorolac after orthopedic surgery. Pain. 1995; 61(2): 291–7. PubMed Abstract \| Publisher Full Text\n\nWhite PF, Way WL, Trevor AJ: Ketamine--its pharmacology and therapeutic uses. Anesthesiology. 1982; 56(2): 119–36. PubMed Abstract \| Publisher Full Text\n\nSubramaniam B, Subramaniam K, Pawar DK, et al.: Preoperative epidural ketamine in combination with morphine does not have a clinically relevant intra- and postoperative opioid-sparing effect. Anesth Analg. 2001; 93(5): 1321–6. PubMed Abstract \| Publisher Full Text\n\nGhorpade A, Advokat C: Evidence of a role for N-methyl d-aspartic acid receptor activation: implications for the treatment of post-injury pain hypersensitivity states. Pain. 1991; 44: 293–9.\n\nReza FM, Zahra F, Esmaeel F, et al.: Preemptive analgesic effect of ketamine in patients undergoing elective cesarean section. Clin J Pain. 2010; 26(3): 223–6. PubMed Abstract \| Publisher Full Text\n\nNgan Kee WD, Khaw KS, Ma ML, et al.: Postoperative analgesic requirement after cesarean section: a comparison of anesthetic induction with ketamine or thiopental. Anesth Analg. 1997; 85(6): 1294–8. PubMed Abstract\n\nSen S, Ozmert G, Aydin ON, et al.: The persisting analgesic effect of low-dose intravenous ketamine after spinal anaesthesia for caesarean section. Eur J Anaesthesiol. 2005; 22(7): 518–23. PubMed Abstract \| Publisher Full Text\n\nMenkiti ID, Desalu I, Kushimo OT: Low-dose intravenous ketamine improves postoperative analgesia after caesarean delivery with spinal bupivacaine in African parturients. Int J Obstet Anesth. 2012; 21(3): 217–21. PubMed Abstract \| Publisher Full Text\n\nBilgen S, Köner O, Türe H, et al.: Effect of three different doses of ketamine prior to general anaesthesia on postoperative pain following Caesarean delivery: a prospective randomized study. Minerva Anestesiol. 2012; 78(4): 442–9. PubMed Abstract\n\nHan SY, Jin HC, Yang WD, et al.: The Effect of Low-dose Ketamine on Post-caesarean Delivery Analgesia after Spinal Anesthesia. Korean J Pain. 2013; 26(3): 270–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLaskowski K, Stirling A, McKay WP, et al.: A systematic review of intravenous ketamine for postoperative analgesia. Can J Anaesth. 2011; 58(10): 911–23. PubMed Abstract \| Publisher Full Text\n\nNistal-Nuño B, Freire-Vila E, Seoane FC, et al.: Data on the effect of preoperative low-dose ketamine in opioid-naïve patients undergoing colon surgery when nitrous oxide is used. F1000Research. 2014. Data Source" }	[ { "id": "6207", "date": "29 Sep 2014", "name": "Arthur Atchabahian", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a useful paper insofar as it seems to indicate that a short-term administration of ketamine does not improve postoperative pain control. The evidence in the literature is contradictory and more data is needed. Study DesignType of surgery and anesthetic technique were homogenous. The exclusion of prior chronic pain syndromes or use of pain medication also helps make the study population more homogeneous, although I would question whether there might be some added benefit of ketamine in patients who are taking chronic opioids.I wish the authors briefly gave the results of their pilot study, especially the standard deviation that was used to calculate the sample size.As the authors say in their discussion, 0.5 mg/kg ketamine is a relatively low dose. With a t 1/2 of 10 -15 min and an active metabolite that has 1/3 efficacy and also a relatively short t 1/2 of 2.5 h, it is questionable whether the objective of modulating the NMDA receptor response was achieved, and that is probably why there was no benefit of the ketamine administration. Maybe the authors should consider repeating the study using a ketamine infusion.Second, the administration of a continuous postoperative morphine infusion of 0.015 mg/kg/h may have created some \"background noise\" and obscured a difference in VAS score or difference in the amount of self-administered morphine. Article contentThe study is well conducted. Data is complete and well analyzed. The discussion is well conducted and addresses the study rationale, findings, and main limitations. There are a few typos: remifentanil, succinylcholine, psychotomimetic.", "responses": [] }, { "id": "7156", "date": "23 Jan 2015", "name": "Lisa V. Doan", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have investigated the role of preoperative ketamine on analgesia in opioid-naive patients undergoing open colon resection. This was a negative study, showing no significant differences in VAS or opioid consumption over the first 24 hours postoperatively. Overall it was a well conducted study. Perhaps the authors could comment on why epidural analgesia was not utilized for open colon resections (though it is unclear whether this would have affected the outcome measures). The dose of ketamine given preoperatively is low. It would be interesting in future studies to use an infusion of ketamine and to include opioid tolerant patients as a study population.", "responses": [] } ]	1	https://f1000research.com/articles/3-226
https://f1000research.com/articles/3-140/v1	01 Jul 14	{ "type": "Software Tool Article", "title": "GASOLINE: a Cytoscape app for multiple local alignment of PPI networks", "authors": [ "Giovanni Micale", "Andrea Continella", "Alfredo Ferro", "Rosalba Giugno", "Alfredo Pulvirenti", "Andrea Continella", "Alfredo Ferro", "Rosalba Giugno" ], "abstract": "Comparing protein interaction networks can reveal interesting patterns of interactions for a specific function or process in distantly related species. In this paper we present GASOLINE, a Cytoscape app for multiple local alignments of PPI (protein-protein interaction) networks. The app is based on the homonymous greedy and stochastic algorithms. To the authors knowledge, it is the first Cytoscape app for computing and visualizing local alignments, without requiring any post-processing operations. GO terms can be easily attached to the aligned proteins for further functional analysis of alignments. GASOLINE can perform the alignment task in few minutes, even for a large number of input networks.", "keywords": [ "In the last few years there has been a rapid growth of biological network data", "including protein-protein interaction (PPI) networks", "metabolic networks and regulatory networks. Among these", "PPI networks are important in several biological phenomena such as signaling", "transcriptional regulation and formation of multi-enzyme complexes1." ], "content": "Introduction\n\nIn the last few years there has been a rapid growth of biological network data, including protein-protein interaction (PPI) networks, metabolic networks and regulatory networks. Among these, PPI networks are important in several biological phenomena such as signaling, transcriptional regulation and formation of multi-enzyme complexes1.\n\nComparing PPI networks of evolutionary distant species can help to understand some mechanisms underlying a specific function or process, which the sequence comparison alone cannot explain. Local network alignment aims to compare networks of different species, in order to find conserved protein complexes or pathways.\n\nIn literature, several network alignment algorithms have been described together with their implementations2–5, however none of them is fully integrated into Cytoscape.\n\nHere, we describe a Cytoscape app implementing the GASOLINE algorithm for multiple local alignment of PPI networks. Aligned proteins can be associated with GO annotations for further functional analysis of alignments. To the authors knowledge, it is the first tool that online computes and visualizes local alignments in a user-friendly way, without requiring any post-processing operations.\n\n\nImplementation\n\nThe GASOLINE app is based on the homonymous greedy and stochastic algorithms introduced in6.\n\nThe app has been written in Java version 7 and designed following a classic Model-View-Controller (MVC) model. The Model part is represented by the classes implementing the algorithm and the auxiliary data structures. The View part is composed by two Java Panels; one for setting all the input and output parameters, and one for listing local alignments and handling their visualization.\n\nThe Controller part ensures the communication between the Model and the View and is implemented by different Cytoscape Task classes, one for each process performed by GASOLINE (i.e. checking file format, computing alignments, importing networks, protein description and GO annotations, building alignment graphs). Each Task class properly notifies the corresponding view class when a task has been completed.\n\nInput networks are imported as text files and then internally represented in two different ways, in order to optimize the performance of our algorithm. We used CyNetwork and CyNetworkView objects for network alignment visualization and custom classes for computing alignments. For all the imported networks, the corresponding Cytoscape view is initially disabled to reduce memory consumption.\n\nThe main component of GASOLINE is represented by a tabbed panel named “GASOLINE”, in the Control Panel of Cytoscape (Figure 1). Through the interface users can provide the following information:\n\n“Similarity information”, to upload orthology similarity scores between proteins of different species\n\n“Networks”, for selecting two or more networks to align\n\n“Parameters setting”, to modify the default GASOLINE parameters\n\n“Optional parameters setting”, for setting other advanced input parameters\n\n“Ontologies”, to upload GO terms linked to the proteins of the aligned networks\n\n“Output”, to specify the folder where the final alignments will be saved\n\nThe button labeled “?”, when present, explains the meaning of a specific function or parameter of GASOLINE, whenever the mouse arrow hovers over that button.\n\nIn the following subsections, we will describe all the required steps to run GASOLINE on a set of PPI networks.\n\nBefore running GASOLINE, the user needs to upload input data, including:\n\na) Two or more networks to be aligned;\n\nb) A file of orthology BLAST bit scores between proteins of different species;\n\nc) A set of GO terms linked to the proteins of each network.\n\nThe GO terms file is not mandatory and can be omitted. Networks are given as a list of weighted edges and can be uploaded from the “Networks” panel.\n\nOrthology data can be uploaded through the “Similarity information” panel. They can be supplied in two different formats: “BLAST Bit scores” or “COG groups”.\n\nThe “BLAST Bit scores” format is a text file where each row has a couple of proteins of different species followed by their corresponding BLAST bit score.\n\nFiles in the “COG groups” associate a list of orthology groups (e.g. KOG, NOG, COG groups) to the proteins of aligning networks. The “COG groups” format can be more convenient when aligning many networks since the all the possible pairwise bit scores are many.\n\nGO categories can be optionally uploaded from “Ontologies” panel. They are provided as text files, where a list of GO cellular components, processes and functions is associated to each protein.\n\nWhenever GO categories are provided as input, the list of GO terms for a specific protein is added as node attribute in Cytoscape, these are accessible from the “Node Browser” tabbed panel once GASOLINE ends the computation and the local alignments are ready to be visualized.\n\nThe main input GASOLINE parameters are specified in the “Parameters setting” panel. These include:\n\n“Iter Seed”: the number of iterations of Gibbs sampling in the bootstrap phase\n\n“Iter Extend”: the number of iterations of Gibbs sampling in each extension step of the iterative phase\n\n“Sigma”: minimum network degree of nodes that can be selected as seeds in the initial phase\n\nValues for “Iter Seed” and “Iter Extend” depend on the number of aligning networks: the more networks we have, the higher these values should be. However, based on the experiments performed on real PPI networks and reported in6, we empirically estabilshed that 200 iterations of Gibbs sampling in both phases are enough to produce reliable results for up to 25 networks. GASOLINE is very fast; it computes the results on 25 networks in a few minutes.\n\nThe choice of “Sigma” implies a tradeoff between speed and accuracy of GASOLINE: the higher the σ, the faster is GASOLINE but the lower its accuracy. If networks are very sparse (like most of the existing PPI networks), low values of sigma (1 or 2) are recommended.\n\nThe “Optional parameters setting” panel contains three more input parameters:\n\n“Overlap”: a value between 0 and 1, denoting the maximum allowed fraction of common nodes between two alignments, in order to be considered distinct. If two alignments have many nodes in common, the one with the least number of nodes is discarded from the final set;\n\n“Refine”: the number of iterations of the GASOLINE iterative phase\n\n“Min Complex Size”: the minimum size of conserved complexes in the final set of local alignments\n\nThese parameters can be modified by checking the box “Active optional settings”, otherwise the default values will be used.\n\nNote that a high value of the “Refine” parameter can be used to increase the accuracy of the local alignments, but the algorithm will be more time consuming. In our tests6, we experienced that a value of 10 guarantees the best trade-off between speed and accuracy of GASOLINE.\n\nFor the “Overlap” and “Min Complex Size” parameters, we suggest 0.5 and 5 as default values, respectively.\n\nFinally, the user can specify an output folder for the final alignments, by clicking on the text field next to the “Output folder” label of the “Output” panel. Each local alignment will be stored in a separate text file inside the specified folder, containing the list of aligned sub-graphs and the one-to-one mapping between aligned nodes.\n\nOnce all the required input files are provided and all the parameters are set up, GASOLINE can be executed by clicking on the “Align” button. Then, a task window will appear describing the progress of the algorithm.\n\nWhen GASOLINE ends, a table containing all the computed local alignments is shown on the right side of the “Results panel” of Cytoscape (Figure 1). The table reports, for each alignment, the size of the aligned complexes and the ISC score.\n\nEach row of the table contains a “Show” button, for the visualization of the corresponding alignment graph on the left side of the “Results Panel” of Cytoscape (Figure 1).\n\nIn the alignment graph, each node is labeled with the ID of the corresponding protein. If GO annotations have been provided, the user can select a node and view the description of the protein and its corresponding GO terms from the “Node Attribute Browser” tabbed panel of Cytoscape.\n\nTwo kinds of edges are shown:\n\nIntra-edge, linking proteins of the same network, which are represented with solid colored lines\n\nInter-edge, linking proteins of different networks that map one another in the local alignment, which are drawn with dashed lines\n\nColors of intra-edges depend on the probability p of the corresponding protein-protein interaction: for low values of p colors range from green to yellow, for high values of p colors range from yellow to red. Weights are automatically associated to edges as attributes, so the user can select an edge and retrieve its weight from the “Edge Attribute Browser” tabbed panel of Cytoscape.\n\nLayout “Kamada-Kawai” has been used for the visualization of the alignment graph.\n\n\nResults\n\nFinally, we show an example of the workflow, using three well known PPI networks C. elegans, D. melanogaster, S. cerevisiae) taken from the STRING database7, considering only experimentally validated interactions. We also annotated proteins by using a set of GO annotations and protein descriptions taken from BioDBnet8.\n\nFollowing the steps described in the Implementation section, we loaded the three networks and ran GASOLINE, using default parameters (IterSeed = 200, IterExtend = 200, Sigma = 7, Overlap = 0.5, Refine = 10, MinComplexSize = 5).\n\nGASOLINE took 110 seconds to complete the task and returned many known conserved complexes with a high degree of topological conservation (ISC between 80 and 90%). These include the large and small subunits of ribosomes (64 proteins), a serine/threonine kinase complex (34), the spliceosome (28), a DNA repair complex (24 proteins, Figure 2), the V-ATPase complex (17) and the ARP2/3 complex (16).\n\n\nConclusions\n\nIn this paper, we presented GASOLINE, an app for Cytoscape 3 for computation and visualization of multiple local alignments of protein-protein interaction networks. To the best of our knowledge, it is the first Cytoscape plugin for computation and visualization of multiple local alignment of biological networks.\n\nGASOLINE offers a user-friendly interface and an easy 2D visualization of local alignments. Moreover, alignments can be further investigated, by attaching GO terms to the proteins of aligning networks.\n\n\nSoftware availability\n\nThe GASOLINE app, as well as datasets of real PPI networks and orthology files that can be directly used to run the algorithm, can be downloaded from the GASOLINE website: http://ferrolab.dmi.unict.it/gasoline/gasoline.html.\n\nThe GASOLINE plugin can also be downloaded from the Cytoscape App Store: http://apps.cytoscape.org/apps/gasoline.\n\nOn our website, there is also a complete documentation on the GASOLINE plugin, with more details about the format of input and output data, and a JAR file for running our algorithm in local with any platform.\n\nLatest source code: https://github.com/GMicale/GASOLINE\n\nSource code as at the time of publication: https://github.com/F1000Research/GASOLINE/releases/tag/V1.0\n\nArchived source code as at the time of publication: http://www.dx.doi.org/10.5281/zenodo.104629", "appendix": "Author contributions\n\n\n\nGM and AP conceived the project. GM and AC designed the architecture of Cytoscape plugin for previous versions of Cytoscape (up to version 2.8). AC wrote a first implementation of the plugin. GM enabled the porting of the code to the new versions of Cytoscape (from version 3.0 onward) and extended the functionality of the application. AP, RG and AF coordinated the project. GM, AP, RG and AF wrote the paper.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBarabasi AL, Oltvai ZN: Network biology: understanding the cell’s functional organization. Nat Rev Genet. 2004; 5(2): 101–113. PubMed Abstract \| Publisher Full Text\n\nFlannick J, Novak A, Srinivasan BS, et al.: Graemlin: general and robust alignment of multiple large interaction networks. Genome Res. 2006; 16(9): 1169–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiao CS, Lu K, Baym M, et al.: IsoRankN: spectral methods for global alignment of multiple protein networks. Bioinformatics. 2009; 25(12): i253–258. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKalaev M, Bafna V, Sharan R: Fast and accurate alignment of multiple protein networks. J Comput Biol. 2009; 16(8): 989–999. PubMed Abstract \| Publisher Full Text\n\nSahraeian SM, Yoon BJ: SMETANA: accurate and scalable algorithm for probabilistic alignment of large-scale biological networks. PLoS One. 2013; 8(7): e67995. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMicale G, Pulvirenti A, Giugno R, et al.: GASOLINE: a Greedy And Stochastic algorithm for Optimal Local multiple alignment of Interaction NETworks. PLoS One. 2014; 9(6): e98750. PubMed Abstract \| Publisher Full Text\n\nSzklarczyk D, Franceschini A, Kuhn M, et al.: The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Res. 2011; 39(Database issue): D561–D568. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMudunuri U, Che A, Yi M, et al.: bioDBnet: the biological database network. Bioinformatics. 2009; 25(4): 555–556. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMicale G, Continella A, Ferro A, et al.: F1000Research/GASOLINE. ZENODO. 2014. Data Source" }	[ { "id": "5404", "date": "10 Jul 2014", "name": "Antonio J. Perez Pulido", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article describes a useful plugin for Cytoscape which can be used to align protein interaction networks, including GO annotations. It is an extension of a previous paper where they presented the algorithm. This fact should be clearly mentioned, and the functionality and use of this kind of analysis should also be showed. The authors should also solve the following points: AbstractThe abstract should better explain what kind of alignments it makes. It could be confused with sequence local alignments. IntroductionWhat does “GASOLINE” mean? It appears on the web site but not in the actual manuscript. “Comparing PPI networks of evolutionary distant species can help to understand some mechanisms”. Indicate what mechanisms. “Local network alignment aims to compare networks of different species, in order to find conserved protein complexes or pathways”. Include reference. The introduction section is too short. Cytoscape should be introduced. What is it, what are the advantages from it and why was it chosen. They should also explain what a network alignment algorithm is. It is a not a usual algorithm. Its utility must be adequately motivated prior to show the new algorithm. In addition, the introduction should show that GASOLINE algorithm was previously published in another article. Is there any difference with the original algorithm? This is a very important issue because the difference with the previous article must be clear. Implementation“the number of iterations of Gibbs sampling in the bootstrap phase / in each extension step of the iterative phase”. Explain what that is. “GASOLINE is very fast; it computes the results on 25 networks in a few minutes”. If it is a result from the previous article, please indicate this or move this to the results. What is the ISC score? “Index of Structural Conservation”. But it is not shown in this manuscript. ResultsThe test took 350 seconds on our computers. Perhaps the authors should indicate what kind of computer was used. Figure 2 should be used to highlight and describe the different panels and information in them. ConclusionsIt should indicate potential and practical uses of GASOLINE to solve biological problems. TyposPlease, review the syntax. For example, “estabilshed” and some English sentences.", "responses": [ { "c_id": "1004", "date": "23 Sep 2014", "name": "Giovanni Micale", "role": "Author Response", "response": "We thank the reviewer for his comments and suggestions. The article describes a useful plugin for Cytoscape which can be used to align protein interaction networks, including GO annotations. It is an extension of a previous paper where they presented the algorithm. This fact should be clearly mentioned, and the functionality and use of this kind of analysis should also be showed. We clearly state that GASOLINE app is an extension of a previous paper and we provided some examples of application of our tool for annotation transfer and protein prediction. In what follows we answer to each question. - ABSTRACT The abstract should better explain what kind of alignments it makes. It could be confused with sequence local alignments. We clarified that GASOLINE has been implemented for solving local network alignment of PPI networks - INTRODUCTION What does “GASOLINE” mean? It appears on the web site but not in the actual manuscript. We defined the acronym for GASOLINE. “Comparing PPI networks of evolutionary distant species can help to understand some mechanisms”. Indicate what mechanisms. We defined what kind of mechanisms (regulation of specific processes of functions within the cell) can be better explained through PPI network alignment. “Local network alignment aims to compare networks of different species, in order to find conserved protein complexes or pathways”. Include reference. We provided references for the most important local alignment methods. The introduction section is too short. Cytoscape should be introduced. What is it, what are the advantages from it and why was it chosen. We added a small paragraph, where we briefly introduced Cytoscape, explaining its advantages. They should also explain what a network alignment algorithm is. It is a not a usual algorithm. Its utility must be adequately motivated prior to show the new algorithm. The network alignment problem has been described more clearly and the differences between local and global alignment methods have been remarked. In addition, the introduction should show that GASOLINE algorithm was previously published in another article. Is there any difference with the original algorithm? This is a very important issue because the difference with the previous article must be clear. In the \"Implementation\" section we clarified that there is no technical difference between GASOLINE Cytoscape app and the original algorithm (for which we provided the reference). - IMPLEMENTATION “the number of iterations of Gibbs sampling in the bootstrap phase / in each extension step of the iterative phase”. Explain what that is. All the GASOLINE parameters have been better described in the \"Introduction\" section, where we provided more details of GASOLINE. We indicated that GASOLINE is based on an iterative Gibbs sampling algorithm, which is applied both in the bootstrap and in the iterative phase (extension steps). “GASOLINE is very fast; it computes the results on 25 networks in a few minutes”. If it is a result from the previous article, please indicate this or move this to the results. This result is taken from the original paper of GASOLINE. However, we decided to remove it from this section and from the whole paper. We specified that GASOLINE is scalable with the number of networks in the “Introduction” section, and we only indicated the running time of GASOLINE for the test reported in the “Results” section.What is the ISC score? “Index of Structural Conservation”. But it is not shown in this manuscript. We defined what the ISC score is and how it is used to evaluate the quality of local alignments. - RESULTS The test took 350 seconds on our computers. Perhaps the authors should indicate what kind of computer was used. We indicated the technical characteristics of the machine used for the tests. Figure 1 should be used to highlight and describe the different panels and information in them. We revised Figure 1 highlighting and describing each GASOLINE panel. - CONCLUSIONSIt should indicate potential and practical uses of GASOLINE to solve biological problems. We give few more details concerning the potential applications of GASOLINE in annotation transfer and protein function prediction. - TYPOSPlease, review the syntax. For example, “estabilshed” and some English sentences.Fixed." } ] }, { "id": "5342", "date": "15 Jul 2014", "name": "Rintaro Saito", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI was unable to install the App GASOLINE to Cytoscape 3.0.In particular, I tried to install GASOLINE to Cytoscape 3.0.2 on Mac OS X 10.9.4 via \"install from file\" and from the App store via App manager. However, I got error messages, and the GASOLINE menu did not appear. I also tried the installation on another machine (Mac OS X snow leopard), but I still got the error.Other Apps such as MCODE or BiNGO could be installed, thus I do not think that it is the problem of my specific environment.Therefore, I would like to request authors to solve this problem.Here is error messages:The task could not be completed because an errorhas occurred.java.lang.Exception: java.lang.UnsupportedClassVersionError: PluginWrapper : Unsupported major.minor version 51.0org.cytoscape.work.internal.task.JDialogTaskManager$TaskRunnable.run(JDialogTaskManager.java:307)java.util.concurrent.Executors$RunnableAdapter.call(Executors.java:439)java.util.concurrent.FutureTask$Sync.innerRun(FutureTask.java:303)java.util.concurrent.FutureTask.run(FutureTask.java:138)java.util.concurrent.ThreadPoolExecutor$Worker.runTask(ThreadPoolExecutor.java:895)java.util.concurrent.ThreadPoolExecutor$Worker.run(ThreadPoolExecutor.java:918)java.lang.Thread.run(Thread.java:695)java.lang.UnsupportedClassVersionError: PluginWrapper : Unsupported major.minor version 51.0java.lang.ClassLoader.defineClass1(Native Method)java.lang.ClassLoader.defineClassCond(ClassLoader.java:637)java.lang.ClassLoader.defineClass(ClassLoader.java:621)java.security.SecureClassLoader.defineClass(SecureClassLoader.java:141)java.net.URLClassLoader.defineClass(URLClassLoader.java:283)java.net.URLClassLoader.access$000(URLClassLoader.java:58)java.net.URLClassLoader$1.run(URLClassLoader.java:197)java.security.AccessController.doPrivileged(Native Method)java.net.URLClassLoader.findClass(URLClassLoader.java:190)java.lang.ClassLoader.loadClass(ClassLoader.java:306)java.lang.ClassLoader.loadClass(ClassLoader.java:247)org.cytoscape.app.internal.manager.SimpleApp.createAppInstance(SimpleApp.java:94)org.cytoscape.app.internal.manager.SimpleApp.install(SimpleApp.java:173)org.cytoscape.app.internal.manager.AppManager$1.onFileCreate(AppManager.java:339)org.apache.commons.io.monitor.FileAlterationObserver.doCreate(FileAlterationObserver.java:379)org.apache.commons.io.monitor.FileAlterationObserver.checkAndNotify(FileAlterationObserver.java:345)org.apache.commons.io.monitor.FileAlterationObserver.checkAndNotify(FileAlterationObserver.java:304)org.cytoscape.app.internal.manager.AppManager.checkForFileChanges(AppManager.java:563)org.cytoscape.app.internal.manager.AppManager.installApp(AppManager.java:631)org.cytoscape.app.internal.task.InstallAppFromFileTask.run(InstallAppFromFileTask.java:36)org.cytoscape.work.internal.task.JDialogTaskManager$TaskRunnable.run(JDialogTaskManager.java:279)java.util.concurrent.Executors$RunnableAdapter.call(Executors.java:439)java.util.concurrent.FutureTask$Sync.innerRun(FutureTask.java:303)java.util.concurrent.FutureTask.run(FutureTask.java:138)java.util.concurrent.ThreadPoolExecutor$Worker.runTask(ThreadPoolExecutor.java:895)java.util.concurrent.ThreadPoolExecutor$Worker.run(ThreadPoolExecutor.java:918)java.lang.Thread.run(Thread.java:695)", "responses": [ { "c_id": "902", "date": "15 Jul 2014", "name": "Giovanni Micale", "role": "Author Response", "response": "The error you encountered was due to the fact that our app was compiled with Java 7, while the last version of Java released by Apple was version 6. So, we recompiled GASOLINE with Java 6 and we tested this new version on all operative systems and it ran without problems. This new version of GASOLINE app (version 1.2) is now available at http://apps.cytoscape.org/apps/gasoline" } ] }, { "id": "5295", "date": "15 Jul 2014", "name": "Byung-Jun Yoon", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper, the authors introduce a Cytoscape plug-in that can be used to perform local alignments of protein-protein interaction (PPI) networks using GASOLINE. The local network algorithm algorithm called GASOLINE was developed by the same team and it was presented in a separate journal paper. As the authors claim, a Cytoscape app for the alignment and visualization of PPI networks may serve as a useful tool for researchers working with multiple PPI networks. However, there are also several issues that need to be properly addressed to make the paper more accessible and informative for potential readers.Abstract:Readers may not be familiar with GASOLINE. Please provide a more detailed description of the algorithm.Introduction: \"In the last few years there has been a rapid growth of biological network data, including protein-protein interaction (PPI) networks, metabolic networks and regulatory networks.\"Please add references for available PPI databases and resources. \"Comparing PPI networks of evolutionary distant species can help to understand some mechanisms underlying a specific function or process, which the sequence comparison alone cannot explain.\"Please provide an example or cite a reference that shows when a simple sequence comparison does not suffice. \"Local network alignment aims to compare networks of different species, in order to find conserved protein complexes or pathways.\"Define network alignment and briefly describe and compare different types of network alignments (e.g. local vs. global). \"In literature, several network alignment algorithms have been described together with their implementations\"At least a brief description of the popular existing methods would be helpful. \"Here, we describe a Cytoscape app implementing the GASOLINE algorithm for multiple local alignment of PPI networks.\"Please provide at least a short introduction to GASOLINE. A brief overview of the algorithm and its pros/cons would be useful.Please explain what type of local alignment is obtained by GASOLINE. (e.g., one-to-one vs. many-to-many).ImplementationDefine acronyms before use (e.g, COG, KOG, NOG, ISC) \"The “COG groups” format can be more convenient when aligning many networks since the all the possible pairwise bit scores are many.\"Meaning unclear. Please revise. \"GO categories can be optionally uploaded from “Ontologies” panel.\"What are the added benefits of uploading GO information? Are they used in any way during the alignment? Does it provide additional information regarding the predicted local alignments? If it does, what kind of extra info can be obtained by uploading GO files? Please describe the capabilities and limitations of the developed Cytoscape app on a typical personal computer. How many networks can be handled by the GASOLINE Cytoscape plug-in? How about the maximum size of the networks? (e.g., number of nodes, number of interactions). How does it scale with the number (size) of networks? Please describe the main difference between the original GASOLINE and the GASOLINE Cytoscape app discussed in this paper. Are there any technical differences? Are there any differences in terms of their capabilities/limitations? Any recommendation on when one should use the desktop version instead of the online version?ConclusionsPlease discuss potential applications of the Cytoscape app.", "responses": [ { "c_id": "1003", "date": "23 Sep 2014", "name": "Giovanni Micale", "role": "Author Response", "response": "We thank the reviewer for his helpful comments and suggestions. - ABSTRACT Readers may not be familiar with GASOLINE. Please provide a more detailed description of the algorithm. We added a paragraph to the Abstract section, providing a more detailed description of GASOLINE. - INTRODUCTION\"In the last few years there has been a rapid growth of biological network data, including protein-protein interaction (PPI) networks, metabolic networks and regulatory networks.\"Please add references for available PPI databases and resources.\"Comparing PPI networks of evolutionary distant species can help to understand some mechanisms underlying a specific function or process, which the sequence comparison alone cannot explain.\"Please provide an example or cite a reference that shows when a simple sequence comparison does not suffice.We added all the references.\"Local network alignment aims to compare networks of different species, in order to find conserved protein complexes or pathways.\"Define network alignment and briefly describe and compare different types of network alignments (e.g. local vs. global).We included a small paragraph describing the network alignment problem and the differences between local and global alignment methods. \"In literature, several network alignment algorithms have been described together with their implementations\"At least a brief description of the popular existing methods would be helpful. We do not give details of most important network alignment methods, but we added references to them. \"Here, we describe a Cytoscape app implementing the GASOLINE algorithm for multiple local alignment of PPI networks.\"Please provide at least a short introduction to GASOLINE. A brief overview of the algorithm and its pros/cons would be useful. Please explain what type of local alignment is obtained by GASOLINE. (e.g., one-to-one vs. many-to-many). We provided more details concerning GASOLINE algorithm and all its phases. Moreover, we added a new paragraph, which highlights the virtues and the current limitations of GASOLINE. In this paragraph we also stress that GASOLINE produces a one-to-one mapping. - IMPLEMENTATION Define acronyms before use (e.g, COG, KOG, NOG, ISC). We defined all the acronyms. \"The “COG groups” format can be more convenient when aligning many networks since the all the possible pairwise bit scores are many.\"Meaning unclear. Please revise. We stressed the benefits of using the \"COG groups\" format in the alignment of many networks. \"GO categories can be optionally uploaded from “Ontologies” panel.\"What are the added benefits of uploading GO information? Are they used in any way during the alignment? Does it provide additional information regarding the predicted local alignments? If it does, what kind of extra info can be obtained by uploading GO files? We clarified that GO information are never used by GASOLINE in the alignment task. They only give, when provided by the user, useful information to better understand the final set of local alignments returned by GASOLINE. Please describe the capabilities and limitations of the developed Cytoscape app on a typical personal computer. How many networks can be handled by the GASOLINE Cytoscape plug-in? How about the maximum size of the networks? (e.g., number of nodes, number of interactions). How does it scale with the number (size) of networks? We specified that theoretically there is no limit on the number and size of input networks. At the end of the “Introduction” section we specified that the running time of GASOLINE is moderately affected by the size and the average density of input networks, but it is scalable with the number of aligning networks.Please describe the main difference between the original GASOLINE and the GASOLINE Cytoscape app discussed in this paper. Are there any technical differences? Are there any differences in terms of their capabilities/limitations? Any recommendation on when one should use the desktop version instead of the online version?At the beginning of this section we underlined that there is no technical difference between the original GASOLINE and the Cytoscape App implementation. The Cytoscape App of GASOLINE wraps the original algorithm and simply includes a visualization module. So overall, the two versions are substantially equivalent. - CONCLUSIONSPlease discuss potential applications of the Cytoscape app.In the last paragraph of this section we sketch some interesting applications of our app, concerning annotation transfer and protein function prediction." } ] } ]	1	https://f1000research.com/articles/3-140
https://f1000research.com/articles/3-163/v1	21 Jul 14	{ "type": "Research Article", "title": "Deletion of ENTPD3 does not impair nucleotide hydrolysis in primary somatosensory neurons or spinal cord", "authors": [ "Eric S. McCoy", "Sarah Street", "Bonnie Taylor-Blake", "Jason Yi", "Martin Edwards", "Mark Wightman", "Mark J. Zylka", "Eric S. McCoy", "Sarah Street", "Bonnie Taylor-Blake", "Jason Yi", "Martin Edwards", "Mark Wightman" ], "abstract": "Ectonucleotidases are membrane-bound or secreted proteins that hydrolyze extracellular nucleotides. Recently, we identified three ectonucleotidases that hydrolyze extracellular adenosine 5’-monophosphate (AMP) to adenosine in primary somatosensory neurons. Currently, it is unclear which ectonucleotidases hydrolyze ATP and ADP in these neurons. Ectonucleoside triphosphate diphosphohydrolases (ENTPDs) comprise a class of enzymes that dephosphorylate extracellular ATP and ADP. Here, we found that ENTPD3 (also known as NTPDase3 or CD39L3) was located in nociceptive and non-nociceptive neurons of the dorsal root ganglion (DRG), in the dorsal horn of the spinal cord, and in free nerve endings in the skin. To determine if ENTPD3 contributes directly to ATP and ADP hydrolysis in these tissues, we generated and characterized an Entpd3 knockout mouse. This mouse lacks ENTPD3 protein in all tissues examined, including the DRG, spinal cord, skin, and bladder. However, DRG and spinal cord tissues from Entpd3-/- mice showed no reduction in histochemical staining when ATP, ADP, AMP, or UTP were used as substrates. Additionally, using fast-scan cyclic voltammetry (FSCV), adenosine production was not impaired in the dorsal spinal cord of Entpd3-/- mice when the substrate ADP was applied. Further, Entpd3-/- mice did not differ in nociceptive behaviors when compared to wild-type mice, although Entpd3-/- mice showed a modest reduction in β-alanine-mediated itch. Taken together, our data indicate that deletion of Entpd3 does not impair ATP or ADP hydrolysis in primary somatosensory neurons or in dorsal spinal cord. Moreover, our data suggest there could be multiple ectonucleotidases that act redundantly to hydrolyze nucleotides in these regions of the nervous system.", "keywords": [ "Nucleotides like ATP are released from neurons and glia throughout the nervous system in response to physiological and pathological stimuli (Arcuino et al.", "2002", "Gourine et al.", "2010", "Matsuka et al.", "2008", "Nakamura & Strittmatter", "1996). Nucleotides signal through activation of the purinergic P2X and P2Y receptors and can excite or sensitize nociceptive neurons (Burnstock", "2007", "Dussor et al.", "2009", "Sawynok", "2007", "Tsuda et al.", "2005). The actions of extracellular ATP can be terminated by several membrane-bound and secreted ectonucleotidases that hydrolyze ATP into adenosine (Sowa et al.", "2010b", "Street et al.", "2013", "Street et al.", "2011", "Vongtau et al.", "2011", "Zimmermann", "2006", "Zylka et al.", "2008). Adenosine", "in turn", "can signal through the A1 adenosine receptor (A1R) to inhibit the activity of nociceptive neurons in the spinal cord (Sawynok & Liu", "2003", "Zylka", "2011)." ], "content": "Introduction\n\nNucleotides like ATP are released from neurons and glia throughout the nervous system in response to physiological and pathological stimuli (Arcuino et al., 2002; Gourine et al., 2010; Matsuka et al., 2008; Nakamura & Strittmatter, 1996). Nucleotides signal through activation of the purinergic P2X and P2Y receptors and can excite or sensitize nociceptive neurons (Burnstock, 2007; Dussor et al., 2009; Sawynok, 2007; Tsuda et al., 2005). The actions of extracellular ATP can be terminated by several membrane-bound and secreted ectonucleotidases that hydrolyze ATP into adenosine (Sowa et al., 2010b; Street et al., 2013; Street et al., 2011; Vongtau et al., 2011; Zimmermann, 2006; Zylka et al., 2008). Adenosine, in turn, can signal through the A1 adenosine receptor (A1R) to inhibit the activity of nociceptive neurons in the spinal cord (Sawynok & Liu, 2003; Zylka, 2011).\n\nWe previously identified and characterized the ectonucleotidases that hydrolyze AMP in nociceptive neurons (Figure 1). These enzymes are Prostatic acid phosphatase (PAP; (Street et al., 2011; Zylka et al., 2008)), Ecto-5’-nucleotidase (NT5E; (Sowa et al., 2010b; Street et al., 2011)), and Tissue-nonspecific alkaline phosphatase (TNAP; (Street et al., 2013)). Pharmacological and knockout mouse model studies suggest that each of these enzymes contributes to the production of adenosine from AMP in the dorsal spinal cord, where nociceptive neurons synapse with spinal neurons (Street et al., 2013). Further, knockout mice lacking PAP, NT5E, or both PAP and NT5E showed enhanced nociceptive sensitization in models of chronic pain (Sowa et al., 2010b; Street et al., 2011; Zylka et al., 2008). While NT5E hydrolyzes AMP into adenosine (optimal activity at neutral pH), PAP (at neutral and acidic pHs) and TNAP (at basic pH) can also hydrolyze ATP, ADP, and AMP (Ciancaglini et al., 2010; Sowa et al., 2009; Street et al., 2013; Zimmermann, 2006). Others found that ectonucleoside triphosphate diphosphohydrolases (ENTPDs), an additional class of ectonucleotidases, might also be responsible for hydrolyzing ATP and ADP in primary somatosensory neurons (Vongtau et al., 2011).\n\nSeveral ectonucleotidases, depicted here, have been shown to hydrolyze adenosine-containing extracellular nucleotides such as ATP in a stepwise process into adenosine.\n\nIn the ENTPD family, four (ENTPD1, -2, -3, and -8) are membrane-bound enzymes that hydrolyze extracellular ATP and ADP (Robson et al., 2006). ENTPD1, -2, and -3 are expressed throughout the central nervous system and display different preferences and kinetics for each nucleotide substrate (Kukulski et al., 2005; Langer et al., 2007). The hydrolysis of ATP by ENTPD1 results in an increase in AMP levels, suggesting ENTPD1 rapidly hydrolyzes ATP and ADP substrates, whereas ENTPD2 preferentially dephosphorylates ATP, resulting in a buildup of extracellular ADP (Figure 1). In contrast, ENTPD3 displays an intermediate activity between ENTPD1 and -2, showing rapid hydrolysis of ATP and transient increases in ADP before conversion into AMP (Kukulski et al., 2005). ENTPD1, -2, and -3 are expressed at similar levels in different cell types of the DRG and spinal cord (Rozisky et al., 2010; Vongtau et al., 2011). Specifically, ENTPD1 is primarily expressed in blood vessels, ENTPD2 is primarily expressed in glial cells, including satellite cells and non-myelinating Schwann cells, and ENTPD3 is preferentially expressed in DRG neurons and their central and peripheral projections (Braun et al., 2004; Vongtau et al., 2011). Further, ENTPD3 co-localizes with markers of nociceptive neurons, such as TRPV1, NT5E, and IB4-binding (Vongtau et al., 2011). These findings suggested that ENTPD3 might contribute to ATP and ADP hydrolysis in nociceptive neurons (Vongtau et al., 2011).\n\nTo study the contribution of ENTPD3 to ATP and ADP hydrolysis in nociceptive and non-nociceptive neurons in the DRG, we generated a knockout mouse that globally lacked ENTPD3 protein. As part of these studies, we performed immunohistochemical experiments to determine which subsets of DRG neurons expressed ENTPD3 and how loss of ENTPD3 altered nucleotide hydrolysis and nociceptive behaviors. Fast-scan cyclic voltammetry (FSCV) was used to examine adenosine generation in wild-type (WT) and Entpd3-/- mice. We found no significant differences between WT and Entpd3-/- mice in assays of ectonucleotidase function or in nociceptive behavioral assays, suggesting that additional enzymes are involved in the hydrolysis of ATP and ADP in nociceptive and non-nociceptive neurons.\n\n\nMethods\n\nAll vertebrate animals and procedures used in this study were approved by the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill. Mice were maintained on a 12 h:12 h light:dark cycle, were given food (Harlan 2920X) and water ad libitum, and were tested during the light phase. Mice were acclimated to the testing room, equipment and experimenter 1–3 days prior to testing.\n\nRecombineering was used to generate the Entpd3 targeting arms from a 129S7/SvEv-derived bacterial artificial chromosome (BAC; bMQ-111o06; CHORI). The start codon, located in exon 2 (Lavoie et al., 2004), was replaced with an AscI site to facilitate cloning of AscI-LoxP-EGFPf-3xpA-LoxP-DTR-pA-Frt-PGK-NeoR-Frt-AscI. EGFPf=farnesylated enhanced GFP (Zylka et al., 2005), DTR=human diptheria toxin receptor (Saito et al., 2001). Use of this construct for axonal tracing and cell ablation of calcitonin gene-related peptide (CGRP)-expressing DRG neurons was previously described (McCoy et al., 2013; McCoy et al., 2012). Correct targeting was confirmed in 5.2% of all embryonic stem cell clones by Southern blotting using flanking 5’ and 3’ probes and a NeoR internal probe. High percentage chimeras were crossed to C57BL/6 females to establish germline transmission and then crossed to PGK1-FLPo mice [B6(C3)-Tg(Pgk1-FLPo)10Sykr/J, Jackson Laboratory] to remove the Frt-flanked selection cassette (confirmed by PCR). Mice were backcrossed to C57BL/6 mice for eight generations to remove the PGK1-FLPo allele (confirmed by PCR) and establish the Entpd3-/- line. Note, the knocked-in GFP was undetectable in DRG and spinal cord neurons of the Entpd3-/- line.\n\nMale WT and Entpd3-/- (3 month-old; ~25 g; n=3 for each genotype) were decapitated, and the DRG and bladder tissue was collected and digested in modified RIPA buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% SDS, 0.5% deoxycholate) supplemented with protease inhibitors (Roche Complete Mini). Protein samples from the cell lysate (1 mg/ml) were analyzed by SDS-PAGE with 4–20% gradient Tris-Glycine polyacrylamide gels (BioRad). The protein samples were then transferred to a polyvinylidene difluoride membrane and probed with a sheep anti-ENTPD3 polyclonal antibody overnight at 4°C (0.2 μg/ml; AF4464, R&D Systems), followed by a rabbit anti-sheep horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature (0.16 μg/ml; #31480, Thermo Scientific).\n\nHindpaw skin (glabrous and hairy), lumbar DRGs, and spinal cords were removed from male mice (n=3; ~10 weeks old) following decapitation, and immersion-fixed in cold 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 3 h, 4 h, and 8 h, respectively, and then cryoprotected in 30% sucrose in 0.1 M phosphate buffer at 4°C. DRGs were sectioned at 20 µm and collected on SuperFrost Plus slides; spinal cords and hindpaw skin were sectioned at 30 µm and 60 µm, respectively, and collected in PBS or a cryoprotectant solution containing PBS, ethylene glycol, and glycerol for long-term storage at -20°C.\n\nEnzyme histochemistry was performed as described previously (Zylka et al., 2008) with a few modifications. Sections of DRG and spinal cord from 3 WT and 3 Entpd3-/- mice were incubated with a given concentration of a nucleotide (AMP, 6 mM for DRG, 3 mM for spinal cord; ADP, 1 mM for DRG and spinal cord; ATP, 0.2 mM for DRGs and spinal cord; UTP, 0.2 mM for spinal cord) in Trizma-maleate buffer containing 20 mM MgCl2, pH 7.0, and 2.4 mM lead nitrate for 3 h at room temperature. For some experiments, we included (in the rinse and substrate incubation steps) 10 mM levamisole to block alkaline phosphatase activity, 5 mM ouabain to block Na+/K+-ATPases, a combination of levamisole and ouabain, or 0.1–1.0 mM ARL67156 (N-diethyl-d-β,γ-dibromomethylene ATP) to nonselectively block ENTPD enzymes. All reagents were purchased from Sigma.\n\nTissue sections from 3 WT and 3 Entpd3-/- mice were stained immunohistochemically as previously reported (Taylor-Blake & Zylka, 2010). Antibodies used were: polyclonal sheep anti-mouse ENTPD3/CD39L3 (skin, 1:75; DRG and spinal cord 1:400; AF4464, R&D Systems), monoclonal mouse anti-NeuN (1:250; MAB377, Millipore), polyclonal chicken anti-Prostatic acid phosphatase (1:4,000; PAP, Aves Labs), polyclonal rabbit anti-NF200 (1:800; N4142, Sigma), monoclonal mouse anti-NF200 (Clone RT97; MAB5262, Millipore), polyclonal rabbit anti-CGRP (1:150; BML-CA1134, Enzo Life Sciences), polyclonal sheep anti-CGRP (1:300; BML-CA1137, Enzo Life Sciences); polyclonal rabbit anti-PKCγ (1:800; sc-211, Santa Cruz), and polyclonal rat anti-PECAM1/CD31 (1:400; Clone MEC 13.3, 553370, BD Biosciences). IB4 conjugated with Alexa Fluor dyes and secondary antibodies conjugated with Alexa Fluor dyes were purchased from Invitrogen. DRAQ5 (Catalog # 4084) was purchased from Cell Signaling Technology. Stained sections were imaged on a Zeiss LSM 510 confocal microscope or a Zeiss LSM 710 confocal microscope.\n\nMouse sagittal spinal cord slices were prepared as described previously (Street et al., 2011). In brief, male mice aged 1–2 months old (~15 g; n=7 for each genotype) were anesthetized with urethane before decapitation, and the spinal cords were dissected and sectioned at 4°C in buffer that contained the following (in mM): 87 NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 75 sucrose, 10 glucose, 1.5 ascorbic acid, 0.5 CaCl2, 7 MgCl2. The slices were then incubated for 45 minutes in artificial cerebrospinal fluid (ACSF), which contained the following (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 25 glucose, 2.5 CaCl2, 1.5 MgCl2. All solutions were bubbled with 95%O2/5%CO2 for the duration of the dissection, incubation, and experiment steps.\n\nFSCV monitoring of adenosine was performed as previously reported (Street et al., 2011), with the major difference being 100 µM ADP was used as the nucleotide substrate. Briefly, a disk-shaped carbon fiber microelectrode (Amoco) was inserted (Cahill et al., 1996), with the disk facing downwards, into the superficial dorsal horn. The potential of the microelectrode was scanned linearly at 400 V/s from -0.4 V to 1.5 V and back again once every 100 ms and was held at -0.4 V otherwise (all potentials versus Ag/AgCl). A micropipette inserted approximately 100 µm from the microelectrode was used to pressure-eject a bolus of 100 µM ADP using a Picospritzer® III (Parker Instrumentation, Pinebrook, NJ) (ejection parameters: 1 s, 20 PSI). The current was recorded for 5 ejections, 5 minutes apart, at the same location in each sample to obtain a mean response. The current was processed, as previously described (Street et al., 2011), using the background subtracted current at the voltammetric peak at ~1.0 V potential, which has been shown to be sensitive to adenosine and not to nucleotides, such as ATP, ADP, and AMP (Swamy & Venton, 2007).\n\nFor all behavioral assays, ~3 month-old male WT (n=10) and Entpd3-/- (n=10; all mice weighing ~26 g) mice were tested in each assay. Mice were acclimated to handling, testing rooms and facilities prior to testing, and the experimenter was blinded to the genotype of each animal. Heat sensitivity was measured by heating each hindpaw once per day using the Plantar Test apparatus (IITC) with a cut-off time of 20 s. For the tail immersion assay, each mouse was gently restrained in a towel, and the distal one-third of the tail was immersed into a water bath heated to 46.5°C or 49°C or into 75% ethanol cooled to -10°C (Wang et al., 1995). The latency to flick or withdraw the tail was measured once per mouse. The cut-off was set at 40 s, 30 s, and 60 s, respectively. For the hot plate test, the latency to jump, shake, or lick a hindpaw was measured within a 30 s cut-off time. To determine mechanical sensitivity, we used an electronic von Frey apparatus (IITC) with semi-flexible tips. Two measurements from each hindpaw were taken and averaged to determine the paw withdrawal threshold in grams. The tail clip assay (noxious mechanical) and cotton swab assay (innocuous mechanical) were performed as described (Garrison et al., 2012; Lariviere et al., 2002). For the acetone test (Bautista et al., 2007), each mouse was placed into a Plexiglas chamber with a wire mesh floor, 50 μL of acetone was placed onto the left hindpaw, and the time spent licking was measured for 1 minute. The cold plantar assay was performed with mice resting on the glass surface of the Plantar Test apparatus (IITC) (Brenner et al., 2012). For the two-temperature discrimination assay, each mouse was placed into a Plexiglas chamber covering two metal surfaces that could be set at different temperatures (Bautista et al., 2007; Dhaka et al., 2007). The amount of time mice spent on each side over a 10 minute period was recorded. Hot and cold sensitivity was assessed on a metal plate heated/cooled to a range of temperatures (5–55°C), with a cut-off time of 30 s, as described (Gentry et al., 2010). For measuring itch responses, histamine (10 μg/μL), chloroquine (CQ; 4 μg/μL) or β-alanine (20 μg/μL) dissolved in 0.9% saline was injected subcutaneously into the nape of the neck (50 μL injection volume). The number of scratching bouts was measured for 30 minutes in 5 minute blocks. One bout consisted of a set of scratches at the injection site until the hindpaw was either licked or placed onto the floor. For the water repulsion assay (Westerberg et al., 2004), the mouse was immersed in a 37°C water bath for 2 min. The mouse was removed from the water and placed onto a paper towel for 5 s, then weight and rectal temperature (deep body temperature, Tb, measured using a digital thermometer, Acorn Temp TC Thermocouple) were measured every 5 min for 60 min. The Complete Freund’s adjuvant (CFA) model of inflammatory pain and the lysophosphatidic acid (LPA) model of neuropathic pain were performed as described (Sowa et al., 2010a; Zylka et al., 2008). Twenty microliters of CFA was injected into the left hindpaw centrally beneath the glabrous skin, and 5 nmol of LPA was administered intrathecally.\n\n\nData analysis\n\nData analysis was performed in Excel (version 2010) using t-tests for all behavioral studies and cell counts with all graphs created in GraphPad Prism. The FSCV data were analyzed using the analysis portion of the freely available software HDCV (Version 4). The software is available for download from: http://www.chem.unc.edu/facilities/index.html?display=electronics&content=software. Average peak currents from the FSCV data were compared using paired t-test. Significance was determined as p ≤ 0.05.\n\n\nResults and discussion\n\nENTPD3 is expressed throughout the nervous system, including nociceptive neurons (Belcher et al., 2006; Langer et al., 2007; Vongtau et al., 2011). To determine which subsets of lumbar DRG neurons expressed ENTPD3, we immunostained for ENTPD3 and markers of nociceptive and non-nociceptive neurons. As previously reported (Vongtau et al., 2011), most DRG neurons, including small-, medium-, and large-diameter neurons, showed some level of staining for ENTPD3 (Figure 2). For colocalization studies, we assessed only those neurons that were stained moderately to strongly for ENTPD3. All cells that expressed ENTPD3 also expressed NeuN, recapitulating previous results showing that ENTPD3 was primarily associated with neuronal cell types (Figure 2A–C, Table 1) (Belcher et al., 2006; Langer et al., 2007; Vongtau et al., 2011). Conversely, 56.8% of all DRG neurons (identified by NeuN expression) labeled for ENTPD3 (Figure 2A–C, Table 1). PAP, a marker of nonpeptidergic and some peptidergic nociceptive neurons, was extensively colocalized with ENTPD3—the majority (72.7%) of DRG neurons expressing PAP also expressed ENTPD3, while almost half (43.5%) of all ENTPD3+ neurons expressed PAP (Figure 2D–F, Table 1). These results were similar to those found by Vongtau and co-workers, who reported that 97% of IB4-binding nonpeptidergic DRG neurons expressed ENTPD3 (Vongtau et al., 2011). NF200, a marker for large-diameter, non-nociceptive neurons and smaller, thinly myelinated (Aδ) nociceptive neurons, colocalized with ENTPD3 (Figure 2G–I, Table 1), suggesting that ENTPD3 was expressed by some non-nociceptive neurons. Finally, an antibody to CGRP was used to identify peptidergic neurons (Figure 2J–L). Of CGRP-expressing neurons, 48.7% were also positive for ENTPD3 (Table 1). Thus, our results indicate that ENTPD3 is expressed in nociceptive and non-nociceptive neurons of the DRG.\n\nMouse DRG neurons were immunostained for ENTPD3 (A,D,G,J) and selected markers (B,E,H,K). (C,F,I,L) Merged images. Images were acquired by confocal microscopy. Scale bar: (in L) A–L = 50 μM.\n\nn=3 animals per genotype; 5 sections per animal. Values represent pooled data from each genotype.\n\nWe also immunostained lumbar spinal cord sections to ascertain where ENTPD3 was located in the dorsal horn, the spinal region where axons of nociceptive and non-nociceptive sensory neurons terminate. ENTPD3+ nerve terminals were located primarily in lamina II, where IB4 terminals are located (Figure 3A–D,I), consistent with a previous report (Vongtau et al., 2011). ENTPD3+ terminals also extended dorsally into lamina I, an area occupied by CGRP+ terminals (Figure 3E,G,I) and ventrally into lamina III, an area with Protein Kinase Cγ (PKCγ)-expressing spinal neurons (Figure 3F,H,J). We also observed small ENTPD3+ spinal neurons in laminae I, II, and III (Figure 3A,B,G,H) as was reported by Vongtau and co-workers (Vongtau et al., 2011). This localization pattern in spinal laminae and spinal neurons suggests that ENTPD3 might hydrolyze extracellular nucleotides in spinal pathways devoted to nociception and somatosensation.\n\nMouse lumbar spinal cord sections were immunostained for ENTPD3 (A–B), and for the indicated markers (C–F). (G–J) Merged images at high (G–H) and low (I–J) magnification. Scale bar in H (A–H) = 50 μM. Scale bar in J (I–J) = 100 μM.\n\nTo assess the extent to which ENTPD3 was necessary for extracellular nucleotide hydrolysis, we disrupted the Entpd3 gene by knocking a LoxP-flanked GFP construct into the start codon of ENTPD3 (Figure 4A). Expression of GFP was not detectable in DRG or spinal cord even when amplified with antibodies against GFP (image not shown). We were thus unable to use GFP to mark cells that expressed Entpd3. Using immunoblotting, we detected ENTPD3 protein in DRG and bladder (tissues known to express high levels of ENTPD3 (Vongtau et al., 2011; Yu et al., 2011)) from WT mice, but no ENTPD3 protein was detectable in tissues from Entpd3-/- mice (Figure 4B). These results confirmed that ENTPD3 protein was eliminated in our knockout line and that the antibody we used was specific for ENTPD3. We also immunohistochemically stained DRG, spinal cord, and hindpaw skin of WT and Entpd3-/- mice. We found that lumbar DRG sections from WT mice showed neuronal staining characteristic of ENTPD3, whereas sections from Entpd3-/- mice showed no staining (Figure 4C,F). Similarly, sections of lumbar spinal cord and hindpaw skin from Entpd3-/- mice showed none of the ENTPD3+ neural profiles observed in WT spinal cord and hindpaw skin (Figure 4D–E,G–H). Mice lacking ENTPD3 produced normal-sized litters (5–9 pups/litter) and had normal weights relative to WT mice (at 3 months ~26 g WT; ~27 g Entpd3-/-).\n\n(A) Cartoon depiction of the farnesylated GFP-DTR construct that was knocked-in to the start codon of Entpd3. (B) Immunoblot showing loss of ENTPD3 protein in DRG and bladder collected from adult mice. (C–H) Loss of ENTPD3 in DRG (F), spinal cord (G), and glabrous skin (H) in Entpd3-/- mouse tissue compared to wild-type tissues (C, D, and E, respectively). Scale bars: D = 50 μM; F = 100 μM; H = 50 μM.\n\nNext, we used immunohistochemistry to determine if primary somatosensory neurons or axon terminals were affected by deletion of Entpd3. In DRG, the number of neurons that expressed nociceptive and non-nociceptive markers was not changed with the exception of a small, but statistically significant decrease in the number of neurons expressing NT5E (Table 2). In WT mice, 35% of DRG neurons expressed NT5E, but in Entpd3-/- animals this percentage was reduced to 30.5% (Table 2). We also used immunohistochemistry to assess whether the spinal dorsal horn of Entpd3-/- mice exhibited altered organization in comparison with that of WT animals. The laminar organization in the dorsal spinal cord of Entpd3-/- mice, as revealed by staining for CGRP and PKCγ and binding of IB4, was indistinguishable from that of WT mice (Figure 5), suggesting that there was no alteration in the organization of primary afferents or spinal neurons in the dorsal horns of mice that lack ENTPD3.\n\nn=3 animals per genotype; 6 sections evaluated per animal. Values represent pooled data from each genotype.\n\nSections of lumbar spinal cord from WT (A) and Entpd3-/- (B) mice were stained with antibodies to distinguish laminar organization of the superficial dorsal horn. Scale bar in B = 100 μM.\n\nFinally, to determine if cutaneous innervation was altered in Entpd3-/- mice, we co-stained sections of glabrous and hairy skin of WT and Entpd3-/- mice with antibodies to ENTPD3 and PGP9.5, a pan-neuronal marker (Figure 6). ENTPD3 marked most PGP9.5+ epidermal free nerve endings in hairy and glabrous skin as well as Meissner corpuscles and Merkel cells in volar pads (Figure 6A–F). These findings were similar to the previously reported staining pattern of ENTPD3 in skin sections (Vongtau et al., 2011). Sections of skin from Entpd3-/- mice lacked all ENTPD3 staining. Expression of PGP9.5 was retained, revealing no differences in the density or structure of free nerve endings, Meissner corpuscles, and Merkel cells in Entpd3-/- mice compared to those observed in skin from WT mice (Figure 6G–L). Thus, cutaneous innervation was not altered by the loss of ENTPD3. Further, nerve fibers co-expressing ENTPD3 and PGP9.5 were found on blood vessels in the dermis and deep dermis of the hindpaw (image not shown). There was no difference in the density of innervation of blood vessels (as revealed by PGP9.5 immunostaining) between WT and Entpd3-/- mice (image not shown). Taken together, these results suggest that, with the exception of a small decrease in NT5E staining in DRG neurons, deletion of Entpd3 did not affect afferents in the skin, DRG neurons, or primary somatosensory afferents in the dorsal spinal cord.\n\nSections of glabrous skin from the volar pads in WT (A–F) and Entpd3-/- (G–L) mice were stained with antibodies against ENTPD3 (A,D,G,J) and PGP9.5 (B,E,H,K). D–F and J–L are high magnification insets of A–C and G–H and show Meissner corpuscles. Scale bar in I (A–C and G–I) = 100 μM. Scale bar in L (D–F and J–L) = 50 μM.\n\nWe previously reported that AMP hydrolysis in the DRG and dorsal spinal cord was redundantly carried out by three ectonucleotidases, PAP, NT5E, and TNAP (Street et al., 2013). However, the enzymes that contribute to ATP and ADP hydrolysis in DRG and spinal cord have not yet been fully characterized. To determine if ENTPD3 contributed to nucleotide hydrolysis in DRG, we performed histochemistry at a neutral pH (7.0) on DRG sections from WT and Entpd3-/- mice using the indicated nucleotides (Figure 7). AMP histochemical staining was found in cell bodies of small- and medium-diameter neurons (Figure 7A,D; where PAP and NT5E are located); ADP histochemical staining was strongest in blood vessels (where ENTPD1 is located) and on the membrane of most neurons (Figure 7B,E); and ATP histochemical staining was present on blood vessels and the cell membrane of most neurons (Figure 7C,F). These staining patterns matched what was previously seen in DRG sections from WT mice (Sowa et al., 2010b; Street et al., 2011; Vongtau et al., 2011; Zylka et al., 2008).\n\nDRG sections from WT (A–C) and Entpd3-/- (D–F) mice were stained using AMP (A,D), ADP (B,E), and ATP (C,F) enzyme histochemistry at pH 7.0 in the presence of 20 mM MgCl2. Concentration of nucleotides used for histochemistry were as follows: AMP, 6 mM; ADP, 1 mM; ATP, 0.1 mM; UTP, 0.1 mM. Scale bar in F = 50 μM.\n\nWhen comparing staining between WT and Entpd3-/- DRGs, we saw no difference in AMP histochemical staining (Figure 7A,D), consistent with the fact that AMP is not a substrate for ENTPD3 (Ciancaglini et al., 2010). Surprisingly however, there were also no differences in histochemical staining between WT and Entpd3-/- DRGs when ADP or ATP was used as substrates (Figure 7B–C,E–F). These data suggest either that ENTPD3 does not hydrolyze these nucleotides in DRG or that other ADP- and ATP-hydrolyzing ectonucleotidases are present and function redundantly with ENTPD3. To determine if ENTPD3 hydrolyzed ADP and ATP redundantly with alkaline phosphatases at pH 7.0, we inhibited alkaline phosphatase activity in histochemical experiments with levamisole (10 mM). However, we observed no difference in staining between WT and Entpd3-/- DRGs in the presence of levamisole (image not shown). These data suggest DRG neurons contain additional ectonucleotidases besides TNAP and ENTPD3 that hydrolyze ATP and ADP at neutral pH.\n\nWe also found that enzyme histochemical staining was equivalent in the superficial dorsal spinal cord of WT and Entpd3-/- mice when the indicated nucleotides were used as substrates (Figure 8). To determine if other enzymes contributed to histochemical staining in the dorsal spinal cord when ATP and UTP (0.2 mM) were used as substrates, we used levamisole to block activity of alkaline phosphatases (10 mM), ouabain to block activity of Na+/K+-ATPase (5 mM), and ARL67156 (0.1 and 1 mM), an inhibitor of ENTPD1 and ENTPD3 (Levesque et al., 2007). The addition of these inhibitors did not result in any change in the staining intensity or pattern in the superficial dorsal horn of WT mice relative to Entpd3-/- mice, but adding ARL67156 caused a near-complete loss of histochemical staining in microglia in the spinal gray in both genotypes, presumably because of blockade of ENTPD1 activity (Braun et al., 2000) (image not shown). Vongtau et al. also tested various inhibitors (ouabain, levamisole, and sodium azide) to block Na+/K+-ATPase, alkaline phosphatase, and ENTPD1 activity, respectively (Vongtau et al., 2011). They found that none of these inhibitors affected ATP or UTP hydrolysis in the spinal cord and concluded that ENTPD3 might be responsible for the remaining staining. Our study demonstrates that the level of nucleotide histochemical staining was the same in the Entpd3-/- mice in the presence of ouabain and levamisole plus an ENTPD1/3 inhibitor (ARL67156), suggesting that one or more enzymes other than ENTPD3 are present that hydrolyze nucleotides in the spinal cord.\n\nLumbar spinal cord sections from WT (A–D) and Entpd3-/- (E–H) mice were stained using AMP (A,E), ADP (B,F), ATP (C,G), and UTP (D,H) enzyme histochemistry at pH 7.0 in the presence of 20 mM MgCl2. Nucleotide concentrations were as follows: AMP, 3 mM; ADP, 1 mM; ATP, 0.2 mM; UTP, 0.2 mM. Scale bar in H = 200 μM.\n\nEnzyme histochemistry detects phosphate that is produced following nucleotide hydrolysis. As an alternative, we used FSCV to quantify adenosine production upon nucleotide hydrolysis in spinal cord slices of WT and Entpd3-/- mice. As previously reported, FSCV can be used to detect adenosine based on characteristic oxidation voltages at 1.0 and 1.5 V (Swamy & Venton, 2007). We applied 100 mM ADP to lamina II and then measured adenosine production at the tip of a carbon-fiber microelectrode (Street et al., 2011). Application of ADP led to the generation of adenosine in WT and Entpd3-/- mice, detected as an increase in measured current at oxidation voltages of 1.0 and 1.5 V (Figure 9A–B). Currents at 1.0 V were then converted to adenosine concentration. We then compared the peak adenosine concentration in WT and Entpd3-/- mice (n=5 slices/genotype) to determine if mice lacking ENTPD3 had any deficit in the production of adenosine (Figure 9C). We saw no significant differences in adenosine generation from ADP between spinal cord slices of WT and Entpd3-/- mice.\n\nFSCV was used to measure adenosine production in response to a bolus of ADP. (A) Representative FSCV color plots: 100 μM of ADP was pressure ejected for 1 s onto lamina II of (A) WT or Entpd3-/- mice. (B) Cyclic voltammogram of one voltage trace from -0.4 V to 1.5 V and back to -0.4 V confirms the production of adenosine in WT (black trace) and Entpd3-/- animals (blue trace) (shown as an increase in current at 1.0 and 1.5 V). (C) Adenosine concentration calculated from 1.0 V current (white horizontal lines in A) in WT (black trace) and Entpd3-/- (blue trace) animals. There was no statistically significant difference between slices from WT and Entpd3-/- mice (n=5 slices for each condition; paired t-test).\n\nThese FSCV results, when combined with enzyme histochemistry results, suggest that there are multiple ectonucleotidases that function redundantly to dephosphorylate ATP and ADP in DRG and superficial dorsal horn. Determining the molecular identities of these enzymes will require future studies with additional ectonucleotidase knockout mice and pharmacological inhibitors. Intriguingly, a redundant group of enzymes mediates AMP hydrolysis in the spinal cord, as PAP, NT5E, and TNAP must all be inhibited to completely block the generation of adenosine from AMP (Street et al., 2013). Likewise, TNAP can fully compensate for the loss of NT5E and generate adenosine from nucleotides in the hippocampus (Zhang et al., 2012).\n\nGiven the high expression of ENTPD3 in nociceptive neurons, we examined whether loss of ENTPD3 affected nociceptive-related behaviors by testing heat, cold, mechanical, and itch sensation (Table 3). In tests of heat sensitivity, there was no difference between WT and Entpd3-/-mice in the tail immersion assay (46.5°C or 49°C; Table 3). Similarly, there was no difference in withdrawal latency in the hot plate test (Table 3). There was also no difference in responses between WT and Entpd3-/- mice in any of the cold assays (acetone evaporative cooling, cold tail immersion at -10°C, or cold plantar; Table 3). To further validate our thermal data, we used a hindpaw withdrawal assay (Gentry et al., 2010) that measures sensitivity to temperatures ranging from noxious cold to noxious hot (Figure 10A). No difference was found between WT and Entpd3-/- mice at any temperature. We also examined responses to mechanical stimuli and observed no difference between WT and Entpd3-/- mice in noxious mechanical (tail clip) and innocuous mechanical (cotton swab) assays (Table 3).\n\nn = 10 mice/group, *p < 0.05.\n\n(A) Sensitivity to temperatures ranging from noxious cold to noxious hot was measured using the hindpaw withdrawal assay. Cutoff time was 30 s. (B) Two-choice temperature discrimination assay. Temperatures were maintained at 25°C/25°C, 25°C/30°C, 20°C/30°C, or 30°C/40°C, and time on each side was measured for 10 minute. (C and D) The water repulsion assay examined rectal body temperature and body weight before and after immersion into a 37°C water bath for 2 min. (E–H) Mechanical allodynia and thermal hyperalgesia were measured in the LPA model of neuropathic pain (E and F) and in the CFA model of inflammatory pain (G and H). n=10 per group. t-tests were used to compare responses between genotypes at each time point, no significant differences detected. All values are represented as means ± SEM.\n\nTo determine if loss of ENTPD3 affected itch, we injected pruritogens (histamine, chloroquine, β-alanine) into the nape of the neck and quantified scratching responses. Histamine- and chloroquine-mediated itch were not altered in Entpd3-/- mice compared to WT mice, but there was a statistically significant reduction (a decrease of 34%) in β-alanine-mediated itch (Table 3). β-alanine activates the Mas-related G-protein-coupled receptor D (MRGPRD) in nonpeptidergic nociceptive neurons (Liu et al., 2012; Rau et al., 2009; Shinohara et al., 2004). Therefore, it is possible that loss of ENTPD3 affects nonpeptidergic DRG neurons. When taken together, these data suggest that ENTPD3 does not play a widespread role in regulating sensitivity to noxious or innocuous somatosensory stimuli.\n\nWe next tested WT and Entpd3-/- mice in a two-temperature discrimination assay. In this assay, the amount of time spent in chambers with equal or different floor temperatures is quantified. Four temperature pairs were evaluated (25°C versus 25°C, 25°C versus 30°C, 20°C versus 30°C, and 30°C versus 40°C). There were no significant differences between WT and Entpd3-/- mice at any of the tested temperature pairs (Figure 10B). These data indicate that temperature discrimination is not impaired in Entpd3-/- mice.\n\nWe next examined the extent to which Entpd3-/- mice regulate body temperature in the water repulsion assay. Mice were placed in a 37°C water bath for 2 minutes and their core body (rectal) temperatures and body weights were measured every 5 minutes for 60 minutes after removal from the water bath (Figure 10C,D). Following removal from the water bath, WT and Entpd3-/- mice showed no differences in the initial body temperature increase or in the subsequent rate to recover their body temperature following hypothermia (Figure 10C). These data demonstrate that Entpd3-/- mice have no deficits in body temperature regulation due to evaporative cooling.\n\nThe water repulsion assay also tests fur barrier function (Westerberg et al., 2004). Once the mouse is removed from the water bath, the initial increase in body weight is indicative of the amount of water absorbed by the fur. We found no significant difference between WT and Entpd3-/- mice in this assay (Figure 10D), including in the rate at which water is removed/evaporates from the mice.\n\nLastly, we sought to determine if deletion of ENTPD3 affected the magnitude of allodynia and hyperalgesia in models of inflammatory pain and neuropathic pain. Lysophosphatidic acid (LPA) is a pronociceptive ligand that sensitizes nociceptors and produces a chemically-induced form of neuropathic pain when injected intrathecally (i.t.) (Inoue et al., 2004). Administration of CFA into the hindpaw causes thermal hyperalgesia and mechanical allodynia and serves as a model of inflammatory pain. We monitored thermal and mechanical sensitivity before and after administration of either LPA (i.t.) or CFA (into hindpaw) and observed no differences between WT and Entpd3-/- mice in either chronic pain model (Figure 10E,F).\n\n\nConclusions\n\nWe generated a mouse that globally lacks ENTPD3 to evaluate the extent to which ENTPD3 was necessary for normal extracellular nucleotide hydrolysis in primary somatosensory neurons and dorsal spinal cord. Despite being expressed at high levels in many nociceptive and non-nociceptive somatosensory neurons, deletion of ENTPD3 did not affect extracellular nucleotide hydrolysis. Further, there were no changes in nociceptive behaviors in Entpd3-/- mice, though we did observe a small reduction in β-alanine-mediated itch response in knockout animals. These findings suggest that other enzymes are present that dephosphorylate extracellular nucleoside di- and triphosphates in primary somatosensory neurons. While ENTPD3 may function redundantly with other ectonucleotidases in these neurons, our Entpd3 knockout line could prove useful in determining the physiological role of ENTPD3 in other organ systems where this ectonucleotidase is expressed, including in neurons that control wakefulness and feeding behavior (Appelbaum et al., 2007; Belcher et al., 2006; Kiss et al., 2009), in the cochlea (Vlajkovic et al., 2006), in cells that regulate insulin secretion (Lavoie et al., 2010; Syed et al., 2013), and in the gastrointestinal system (Lavoie et al., 2011).\n\n\nData availability\n\nF1000Research: Dataset 1. Influence of ENTPD3 deletion on nucleotide hydrolysis in mouse primary somatosensory neurons and spinal cord: data, http://dx.doi.org/10.5256/f1000research.4563.d31211 (McCoy et al., 2014).", "appendix": "Author contributions\n\n\n\nESM performed behavioral experiments, maintained the knockout line, and helped to draft the manuscript. SES helped to generate the Entpd3-/- mouse line, maintained the line, assisted with FSCV experiments, and helped to draft the manuscript. BTB performed enzyme histochemical and immunohistochemical experiments. JJY performed western blot experiments. MAE and RMW assisted with FSCV experiments. MJZ conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by grants to MJZ from NINDS (R01NS067688) and a grant to RMW from the NIH (R01NS038879). The Molecular Neuroscience Core and the Confocal and Multiphoton Imaging Core, where imaging work was performed, are funded by grants from NINDS (P30NS045892) and NICHD (P30HD03110).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript\n\n\nAcknowledgments\n\nWe would like to thank JrGang Cheng and the Molecular Neuroscience Core at the UNC Neuroscience Center for generating the BAC targeting clone and Gabriela Salazar for technical assistance and help with managing the mouse colony.\n\n\nReferences\n\nAppelbaum L, Skariah G, Mourrain P, et al.: Comparative expression of p2x receptors and ecto-nucleoside triphosphate diphosphohydrolase 3 in hypocretin and sensory neurons in zebrafish. Brain Res. 2007; 1174: 66–75. PubMed Abstract \| Publisher Full Text\n\nArcuino G, Lin JH, Takano T, et al.: Intercellular calcium signaling mediated by point-source burst release of ATP. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSowa NA, Taylor-Blake B, Zylka MJ: Ecto-5'-nucleotidase (CD73) inhibits nociception by hydrolyzing AMP to adenosine in nociceptive circuits. J Neurosci. 2010; 30(6): 2235–2244. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSowa NA, Vadakkan KI, Zylka MJ: Recombinant mouse PAP has pH-dependent ectonucleotidase activity and acts through A(1)-adenosine receptors to mediate antinociception. PLoS One. 2009; 4(1): e4248. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStreet SE, Kramer NJ, Walsh PL, et al.: Tissue-nonspecific alkaline phosphatase acts redundantly with PAP and NT5E to generate adenosine in the dorsal spinal cord. J Neurosci. 2013; 33(27): 11314–11322. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStreet SE, Walsh PL, Sowa NA, et al.: PAP and NT5E inhibit nociceptive neurotransmission by rapidly hydrolyzing nucleotides to adenosine. Mol Pain. 2011; 7: 80. 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PubMed Abstract \| Publisher Full Text\n\nVlajkovic SM, Vinayagamoorthy A, Thorne PR, et al.: Noise-induced up-regulation of NTPDase3 expression in the rat cochlea: Implications for auditory transmission and cochlear protection. Brain Res. 2006; 1104(1): 55–63. PubMed Abstract \| Publisher Full Text\n\nVongtau HO, Lavoie EG, Sevigny J, et al.: Distribution of ecto-nucleotidases in mouse sensory circuits suggests roles for nucleoside triphosphate diphosphohydrolase-3 in nociception and mechanoreception. Neuroscience. 2011; 193: 387–398. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang JJ, Ho ST, Hu OY, et al.: An innovative cold tail-flick test: the cold ethanol tail-flick test. Anesth Analg. 1995; 80(1): 102–107. PubMed Abstract\n\nWesterberg R, Tvrdik P, Undén AB, et al.: Role for ELOVL3 and fatty acid chain length in development of hair and skin function. J Biol Chem. 2004; 279(7): 5621–5629. PubMed Abstract \| Publisher Full Text\n\nYu W, Robson SC, Hill WG: Expression and distribution of ectonucleotidases in mouse urinary bladder. PLoS One. 2011; 6(4): e18704. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang D, Xiong W, Chu S, et al.: Inhibition of hippocampal synaptic activity by ATP hypoxia or oxygen-glucose deprivation does not require CD73. PLoS One. 2012; 7(6): e39772. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZimmermann H: Ectonucleotidases in the nervous system. Novartis Found Symp. 2006; 276: 113–128; discussion 128–130, 233–237, 275–281. PubMed Abstract\n\nZylka MJ: Pain-relieving prospects for adenosine receptors and ectonucleotidases. Trends Mol Med. 2011; 17(4): 188–196. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZylka MJ, Rice FL, Anderson DJ: Topographically distinct epidermal nociceptive circuits revealed by axonal tracers targeted to Mrgprd. Neuron. 2005; 45(1): 17–25. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "5504", "date": "01 Aug 2014", "name": "Terry Kirley", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper by McCoy at al. entitled “Deletion of ENTPD3 does not impair nucleotide hydrolysis in primary somatosensory neurons or spinal cord” is a detailed and technically sound study. This paper focuses on possible roles for ENTPD3/NTPDase3 in pain perception and neural transmission in the dorsal root ganglia (DRG) and spinal cord. In addition, other proteins of interest that are related and known to be involved in the DRG, including prosthetic acid phosphatase (PAP), 5’-nucleotidase (NT5E) and tissue nonspecific acid phosphatase (TNAP) were examined. Many behavioral and pain assays were performed. These include tail immersion assays, a hot plate test, tail clip assay, cotton swab assay, acetone test, cold plantar assay, and 2-temperature discrimination assays. In addition, several assays for itch were performed, including histamine, chloroquine, and beta-alanine induced itch. Also, several other behavioral assays, including the water repulsion assay, the Complete Freund’s Adjuvant (CFA) inflammation pain assay and the LysoPhosphatidic Acid (LPA) neuropathic pain assays were performed. In general, the work is well done and detailed, and includes the appropriate controls. However, the main problem with this work is that there is no indication of the physiological function of NTPDase3/ENTPD3 revealed by any of the experimental results. Of course, negative data is sometimes important, and this is so in this case. However, what is really missing from this work is analysis for other nucleotidases that are likely to compensate for the loss of NTPDase3 in these mice. These include the nucleotide pyrophosphatase/phosphodiesterase enzymes (NPPs), and more importantly, the other members of the cell-surface NTPDase class of nucleotidases, especially NTPDase1/ENTPD1, and NTPDase2/ENTPD2. Since the authors claim in their abstract that “there could be multiple ectonucleotidases that act redundantly to hydrolyze nucleotides in these regions of the nervous system” (which seems logical and likely), it is somewhat curious that no analyses for these other nucleotidases were performed. If such experiments were done, and if up-regulation of one or more of these enzymes was observed, this study would be more interesting, and the paper more important, since this might suggest putative physiological role(s) for NTPDase3/ENTPD1. There are a couple of interesting and possibly problematic experimental details reported in the paper. First, why was 20 mM magnesium chloride used in the enzyme histochemistry experiments? This seems to be an unreasonably high, non-physiologic, concentration of magnesium. In addition, many of these nucleotidases, including NTPDase3 and other NTPDases, are, in fact, more active using calcium as a divalent cation as opposed to magnesium. So the choice of 20 mM magnesium chloride seems odd. Also, one change in these knockout mice that is noted in terms of possible effects on nucleotide hydrolysis is the decrease in 5’-nucleotidase protein seen in the DRG neurons of the knockout mouse, which is reported in Table 2. However, as reported in Figure 7, there is no apparent decrease in hydrolysis of AMP in the same dorsal root ganglia, which is apparently not consistent with Table 2, although other enzymes could come into play (but don’t seem to change in the KO). However, enzyme histochemistry is difficult to accurately quantitate and is usually regarded as a semi-quantitative technique. Thus, a relatively small change in hydrolysis rates may not be evident from enzyme histochemical data. This is a potential problem with Figure 7, and begs the question as to why tissue homogenates were not evaluated by solution-based, quantitative nucleotidase enzyme assays. The same limitations are applicable to the data reported in Figure 8 on spinal cord sections from wild type and knockout mice. As reported in Table 3, the authors did find a significant difference in itch response to beta-alanine in the KO mice. However, again, the other itch data, and the rest of the data in Table 3, show no difference between wt and KO mice for responses to itch, heat, or cold behavioral stimuli. In their conclusion section, the authors do mention other roles that have been suggested for NTPDase3/ENTPD3, including possible roles in the hypothalamus for controlling wakefulness and feeding behavior, for hearing in the cochlea, in the beta cells of the pancreas for regulation of ATP-controlled insulin secretion, and in the G.I. tract. It would be interesting to report any experiments designed to monitor for changes in any of these putative physiological functions of NTPDase3. These could include measurements designed to detect abnormal sleep times or cycles, abnormal eating habits, and abnormal plasma glucose and insulin levels and/or abnormal responses to glucose tolerance tests. In conclusion, this study is well done and thorough with respect to the attributes that were evaluated in the DRG and spinal cord. Unfortunately, the results do not suggest likely physiological function(s) for NTPDase3/ENTPD3. Also, there is no data reported for other related cell-surface nucleotidases, such as NTPDases 1 and 2, which may be up-regulated in a compensatory response to the knockout of NTPDase3. In addition, there is no mention of experiments designed to address other putative physiological functions of NTPDase3, which are not related to the DRG or spinal cord. Hopefully, these points will be addressed in future work on these knockout animals.", "responses": [ { "c_id": "979", "date": "11 Sep 2014", "name": "Mark Zylka", "role": "Author Response", "response": "We thank Dr. Kirley for his comments and for appreciating that our study, which is focused on Entpd3 in sensory neurons and spinal cord, largely reports negative findings. This marks the first reported Entpd3 knockout mouse. A priori, it was unknown if this ectonucleotidase would affect nucleotide hydrolysis in sensory neurons or spinal cord independent of the other ectonucleotidases that are present in these tissues. Since deletion of Entpd3 had no effect on nucleotide hydrolysis, we performed additional analyses to determine if other ectonucleotidases were involved. This includes assessing whether alkaline phosphatases act redundantly with Entpd3 (using the inhibitor levamisole), assessing whether other Entpds act redundantly with Entpd3 (using the nonselective Entpd inhibitor ARL67156), and assessing whether Na/K-ATPases act redundantly with Entpd3 (using ouabain to inhibit these ATPases). The outcomes of these experiments are described in the results section.Drs. Kirley and Molliver raised the question of whether other ectonucleotidases might be upregulated and compensate for the loss of ENTPD3. We understand the rationale and interest behind this comment. At this time, we feel such experiments are beyond the scope and focus of our initial study for the following reasons:If we were to find that other ecto-enzymes were upregulated, this information alone would not prove that any upregulated enzymes compensate or act redundantly in the absence of Entpd3. There are numerous ectonucleotidases (NTPDases, NPPs, alkaline phosphatases, acid phosphatases, Na/K-ATPases and likely additional enzymes with ectonucleotidase activity) that could be altered upon Entpd3 deletion. We thus may miss the most relevant enzyme (or enzymes) if we focus on a candidate list. Indeed, our attempt to focus on several candidates, via our use of inhibitors, was unsuccessful, suggesting this is a complex issue with multiple enzymes involved. A genome-wide expression analysis would be more comprehensive but would not provide insights as to which upregulated genes are biologically relevant—knowing what genes change will not allow us to prove they compensate for the loss of Entpd3. Our study was not focused on examining potential compensatory mechanisms in the Enptd3-/- mice. Such a study will require substantial effort to do properly (i.e. we would first need to identify all the enzymes that are upregulated and then demonstrate, in a systematic manner, that each one does or does not act redundantly using inhibitors and/or double/triple knockout mice). Such an endeavor would require considerable effort. For example, it took us several years and multiple knockout lines to rigorously demonstrate that PAP, NT5E and TNAP act redundantly to generate adenosine from AMP. Thus, in lieu of examining a candidate list of ectonucleotidases, we felt a compromise would be to address this comment as follows (by revising the Conclusions section): “Our use of inhibitors ruled out the possibility that some ENTPDs, alkaline phosphatases and Na/K-ATPase compensated for the loss of ENTPD3. However, we cannot exclude the possibility that additional known or unknown enzymes with ectonucleotidase activity might be upregulated in Entpd3-/- mice and compensate for the loss of ENTPD3. Determining which enzymes act redundantly with ENTPD3 will require use of additional inhibitors and additional ectonucleotidase knockout lines.”Dr Kirley: \"There are a couple of interesting and possibly problematic experimental details reported in the paper. First, why was 20 mM magnesium chloride used in the enzyme histochemistry experiments? This seems to be an unreasonably high, non-physiologic, concentration of magnesium. In addition, many of these nucleotidases, including NTPDase3 and other NTPDases, are, in fact, more active using calcium as a divalent cation as opposed to magnesium. So the choice of 20 mM magnesium chloride seems odd.\"To address this comment, we performed new experiments. We performed histochemistry experiments with 2 mM CaCl2, 20 mM CaCl2 and 20 mM MgCl2, in WT and Entpd3-/- mice. These data are shown in new Figure 9.We also updated the results to include this new information:“Many ectonucleotidases, including ENTPD3 (Lavoie et al reference: PMID: 15130768; currently ref #23 “Cloning and characterization of mouse nucleoside triphosphate diphosphohydrolase-3”), are slightly more active in biochemical assays with calcium as the divalent cation. However, we detected no difference in UTP histochemical activity in spinal cord between WT and Entpd3-/- mice when 2 mM or 20 mM CaCl2 was substituted for 20 mM MgCl2 (Figure 9; with deletion of ENTPD3 confirmed in these sections using immunostaining, Figure 9H). Thus Mg2+ and Ca2+ appear to be interchangeable in this histochemical assay.” Also please note, we used 20 mM MgCl in our histochemical experiments because a previous study, which we based our histochemistry method on, found that ATP ectonucleotidase activity in skin Langerhans cells was divavelent cation dependent, with complete interchangability between Ca2+ and Mg2+, and with optimal staining at a 20 mM concentration of either divalent (see Chaker et al., 1984). 20 mM MgCl2 or 20 mM CaCl2 thus appears to be optimal for ATP histochemical staining. And in biochemical assays with ENTPDs, Mg2+ and Ca2+ were interchangeable when ATP and ADP were used as substrates (Rucker et al., 2008). Dr Kirley: \"Also, one change in these knockout mice that is noted in terms of possible effects on nucleotide hydrolysis is the decrease in 5’-nucleotidase protein seen in the DRG neurons of the knockout mouse, which is reported in Table 2. However, as reported in Figure 7, there is no apparent decrease in hydrolysis of AMP in the same dorsal root ganglia, which is apparently not consistent with Table 2, although other enzymes could come into play (but don’t seem to change in the KO). However, enzyme histochemistry is difficult to accurately quantitate and is usually regarded as a semi-quantitative technique. Thus, a relatively small change in hydrolysis rates may not be evident from enzyme histochemical data. This is a potential problem with Figure 7, and begs the question as to why tissue homogenates were not evaluated by solution-based, quantitative nucleotidase enzyme assays. The same limitations are applicable to the data reported in Figure 8 on spinal cord sections from wild type and knockout mice.\"We agree that histochemical staining provides a semi-quantitative readout of enzyme activity. This is why we turned to FSCV in spinal cord slices. FSCV provides a quantitative electrochemical method for measuring hydrolysis of ADP to adenosine, in the precise anatomical region where Entpd3 is located. Since we found no differences between WT and Entpd3-/- mice using this quantitative electrochemical technique, we feel these data are sufficient to show that loss of Entpd3 alone has no measurable effect on nucleotide hydrolysis. And, as can be seen from our micrographs, Entpd3 is restricted to the dorsal spinal cord while ATP and ADP histochemical activity is broadly distributed. Use of a solution-based assay would entail creating homogenates from spinal cord or DRG, thus disrupting the integrity of the tissue and introducing more ectonucleotidases into the assay (which would reduce signal-to-noise). The small 4.5% reduction in NT5E in DRG was statistically significant, although it appears to have no effect on AMP hydrolysis, as assessed histochemically. This likely reflects that AMP can be hydrolyzed by NT5E, PAP and TNAP, as we previously found. Since this did not constitute a major finding, we did not focus or discuss this in the text. Dr Kirley: \"As reported in Table 3, the authors did find a significant difference in itch response to beta-alanine in the KO mice. However, again, the other itch data, and the rest of the data in Table 3, show no difference between wt and KO mice for responses to itch, heat, or cold behavioral stimuli.\" We felt it would be difficult to experimentally pursue the mechanistic basis for this itch phenotype because it was extremely small in magnitude. Such small behavioral effects are not easy to pursue. Moreover, it was the only sensory phenotype out of a large number of sensory functions we probed, suggesting it is a very mild sensory phenotype.Dr Kirley: \"In their conclusion section, the authors do mention other roles that have been suggested for NTPDase3/ENTPD3, including possible roles in the hypothalamus for controlling wakefulness and feeding behavior, for hearing in the cochlea, in the beta cells of the pancreas for regulation of ATP-controlled insulin secretion, and in the G.I. tract. It would be interesting to report any experiments designed to monitor for changes in any of these putative physiological functions of NTPDase3. These could include measurements designed to detect abnormal sleep times or cycles, abnormal eating habits, and abnormal plasma glucose and insulin levels and/or abnormal responses to glucose tolerance tests.\"These are indeed interesting topics for future study. However, we feel they are beyond scope of our present study, which is focused on examining the function of Entpd3 in primary somatosensory neurons and dorsal spinal cord. Dr Kirley: \"In conclusion, this study is well done and thorough with respect to the attributes that were evaluated in the DRG and spinal cord. Unfortunately, the results do not suggest likely physiological function(s) for NTPDase3/ENTPD3. Also, there is no data reported for other related cell-surface nucleotidases, such as NTPDases 1 and 2, which may be up-regulated in a compensatory response to the knockout of NTPDase3. In addition, there is no mention of experiments designed to address other putative physiological functions of NTPDase3, which are not related to the DRG or spinal cord. Hopefully, these points will be addressed in future work on these knockout animals.\"We agree that future studies are warranted. By reporting our findings with these first ever Entpd3 knockout mice, it will now be possible for us and others to study Entpd3 in other physiological contexts and to explore possible compensatory mechanisms." } ] }, { "id": "5940", "date": "28 Aug 2014", "name": "Derek Molliver", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper, McCoy et al. present novel results regarding the contribution of ENTPD3 to nucleotide hydrolysis in primary sensory neurons and the spinal cord. Immunohistochemical analysis of ENTPD3 distribution using a commercial antibody was highly similar to a previous report (full disclosure, from this reviewer’s laboratory) cited in the paper; selectivity of the antibody was effectively confirmed using tissue from knockout mice generated by the authors. The study is well-executed and the figures are outstanding. The key findings were generated through functional analysis of the knockout mice, including nucleotidase histochemistry, extensive behavioral analysis of nociceptive and non-nociceptive thresholds in naïve and inflamed mice, and fast-scan cyclic voltammetry (FSCV), an innovative method for measuring adenosine levels in situ in spinal cord slices. These studies found that ENTPD3 was dispensible for nucleotide hydrolysis, and all measured behavioral variables were unaltered in knockout mice, with the exception of a reduction in b-alanine-mediated itch behavior. The results suggest the possibility that additional ectonucleotidase(s) are co-expressed with ENTPD3 that are sufficient for normal nucleotide triphosphate/diphosphate hydrolysis, similar to the situation described by these authors for the ectonucleotidases that generate adenosine from AMP. The histological results support the conclusion that ENTPD3 is in position to impact both noxious and non-noxious somosensory transduction and transmission, but underscore that the regulation of somatosensory purinergic signaling is complex and likely to be regulated by multiple enzymes acting in tandem. This is a significant finding for the pain field, because ENTPD3 is the only ectonucleotidase identified in primary sensory neurons that regulates the availability of extracellular ATP and UTP, which have been extensively implicated in nociceptive signaling as agonists for P2X and P2Y receptors. A key point not fully addressed here is whether knockout of ENTPD3 results in upregulation of other NTPDases in DRG neurons and/or dorsal horn, which could provide an explanation for the mild knockout phenotype. In particular, analysis of neuronal mRNA/ protein levels and distribution for ENTPD1, 2 and 8 in ENTPD3 knockout mice would increase the impact of the findings reported here. An intriguing possibility is that the alternate enzymes responsible are not members of the ENTPD family. The authors do demonstrate that the ENTPD1 inhibitor ARL67156 did not alter the distribution of enzyme histochemical staining in knockout tissue compared to WT, but did eliminate microglial labeling in both genotypes. One question that the authors might want to address in the discussion is how to evaluate whether FSCV is capable of resolving neuronal ENTPD3 activity in the dorsal horn when the neurons are surrounded by microglia expressing ENTPD1 (the active site of these enzymes is extracellular). As the authors suggest, further analysis in mice with multiple ENTPD gene deletions may be informative. However, the substantial behavioral evidence indicates that loss of ENTPD3 is not critical for normal sensory processing.", "responses": [ { "c_id": "978", "date": "11 Sep 2014", "name": "Mark Zylka", "role": "Author Response", "response": "We thank Dr. Molliver for his comments, particularly given his expertise in studying ENTPD3 in sensory neurons. As Dr. Molliver notes, we attempted to address this issue of compensation using a number of inhibitors in combination with Entpd3-/- tissues.Drs. Kirley and Molliver raised the question of whether other ectonucleotidases might be upregulated and compensate for the loss of ENTPD3. We understand the rationale and interest behind this comment. At this time, we feel such experiments are beyond the scope and focus of our initial study for the following reasons:If we were to find that other ecto-enzymes were upregulated, this information alone would not prove that any upregulated enzymes compensate or act redundantly in the absence of Entpd3. There are numerous ectonucleotidases (NTPDases, NPPs, alkaline phosphatases, acid phosphatases, Na/K-ATPases and likely additional enzymes with ectonucleotidase activity) that could be altered upon Entpd3 deletion. We thus may miss the most relevant enzyme (or enzymes) if we focus on a candidate list. Indeed, our attempt to focus on several candidates, via our use of inhibitors, was unsuccessful, suggesting this is a complex issue with multiple enzymes involved. A genome-wide expression analysis would be more comprehensive but would not provide insights as to which upregulated genes are biologically relevant—knowing what genes change will not allow us to prove they compensate for the loss of Entpd3. Our study was not focused on examining potential compensatory mechanisms in the Enptd3-/- mice. Such a study will require substantial effort to do properly (i.e. we would first need to identify all the enzymes that are upregulated and then demonstrate, in a systematic manner, that each one does or does not act redundantly using inhibitors and/or double/triple knockout mice). Such an endeavor would require considerable effort. Thus, in lieu of examining a candidate list of ectonucleotidases, we felt a compromise would be to address this comment as follows (by revising the Conclusions section):“Our use of inhibitors ruled out the possibility that some ENTPDs, alkaline phosphatases and Na/K-ATPase compensated for the loss of ENTPD3. However, we cannot exclude the possibility that additional known or unknown enzymes with ectonucleotidase activity might be upregulated in Entpd3-/- mice and compensate for the loss of ENTPD3. Determining which enzymes act redundantly with ENTPD3 will require use of additional inhibitors and additional ectonucleotidase knockout lines.” Dr Molliver: \"One question that the authors might want to address in the discussion is how to evaluate whether FSCV is capable of resolving neuronal ENTPD3 activity in the dorsal horn when the neurons are surrounded by microglia expressing ENTPD1 (the active site of these enzymes is extracellular). As the authors suggest, further analysis in mice with multiple ENTPD gene deletions may be informative. However, the substantial behavioral evidence indicates that loss of ENTPD3 is not critical for normal sensory processing.\" To address this comment, we added the following sentence to the Results & Discussion section:“Note that FSCV cannot resolve neuronal ENTPD3 activity in the dorsal horn from spinal microglial ENTPD1 activity, so the adenosine detected by FSCV after applying ADP could originate from microglial ENTPD1 or other ectonucleotidases in the tissue. For example, this adenosine could originate from PAP and/or TNAP, as these enzymes are located in the same region and can also hydrolyze ADP to adenosine (Figure 1).”" } ] } ]	1	https://f1000research.com/articles/3-163
https://f1000research.com/articles/3-175/v1	30 Jul 14	{ "type": "Software Tool Article", "title": "shinyMethyl: interactive quality control of Illumina 450k DNA methylation arrays in R", "authors": [ "Jean-Philippe Fortin", "Elana Fertig", "Kasper Hansen", "Elana Fertig" ], "abstract": "We present shinyMethyl, a Bioconductor package for interactive quality control of DNA methylation data from Illumina 450k arrays. The package summarizes 450k experiments into small exportable R objects from which an interactive interface is launched. Reactive plots allow fast and intuitive quality control assessment of the samples. In addition, exploration of the phenotypic associations is possible through coloring and principal component analysis. Altogether, the package makes it easy to perform quality assessment of large-scale methylation datasets, such as epigenome-wide association studies or the datasets available through The Cancer Genome Atlas portal. The shinyMethyl package is implemented in R and available via Bioconductor. Its development repository is at https://github.com/jfortin1/shinyMethyl.", "keywords": [ "The recent release of the R package shiny1 has substantially lowered the barriers to interactive visualization in R", "opening the door to interactive exploration of high-dimensional genomic data." ], "content": "Introduction\n\nThe recent release of the R package shiny1 has substantially lowered the barriers to interactive visualization in R, opening the door to interactive exploration of high-dimensional genomic data.\n\nDNA methylation is an epigenetic mark, and changes in DNA methylation have been associated with various diseases, such as cancer2. For DNA methylation data, thousands of samples from the state-of-the-art Illumina 450k methylation array3 have been generated and are accessible online from The Cancer Genome Atlas (TCGA) and through the Gene Expression Omnibus (GEO). This array has a series of probes used to measure a methylation and an unmethylation signal for a series of loci. Probes are designed using two main chemistries resulting in a challenging array design, essentially a mix of a two color and a one color array discussed in Bibikova et al.3. Analysis of data from this array requires careful quality control and pre-processing that account for these distinct chemistries. The assessment of these steps could benefit from an interactive visualization tool.\n\nOur solution is shinyMethyl, an interactive visualization package for 450k arrays, based on the packages minfi4 and shiny1. The goal of shinyMethyl is two-fold; (1) to help with quality assessment and (2) to help with assessing the effect of pre-processing. We use pre-computation to enable interactive visualization of thousands of samples to circumvent computational bottlenecks during data exploration. The pre-computation can happen on a large computing server and the resulting data object can be used for interactive visualization on a laptop. Quality control and pre-processing large 450k datasets become easy and intuitive with shinyMethyl.\n\n\nMethods\n\nThe first step of shinyMethyl is pre-computation of various summaries of the 450k array data, using the function shinySummarize. This pre-computation is run on raw (not pre-processed) data and – optionally – pre-processed data, resulting in either one or two summary objects, as described below. These summary objects, called shinyMethylSet, are saved in a platform-independent format. The interactive interface is then launched via the function runShinyMethyl. The function requires a shinyMethylSet containing the summary data from the raw data. In addition, the function accepts as a second argument a shinyMethylSet that contains summaries from pre-processed data, in which case both raw and pre-processed data will be displayed in the interactive interface. Figure 1 illustrates the shinyMethyl workflow.\n\nIDAT files are parsed using minfi and illuminaio into a RGChannelSet. This object is summarized using shinySummarize. Optionally, the data are pre-processed and the pre-processed data are summarized. For visualization, runShinyMethyl is used on either one or two sets of summarized data.\n\nSummarizing the raw data uses the minfi4 and illuminaio5 R packages to parse Illumina IDAT files into a minfi object called RGChannelSet. shinySummarize operates on this RGChannelSet and the summarization object created by this function is 35x smaller than the full data representation in minfi; 1,000 samples use 205 MB. Specifically, the summarized data contain the quantile distributions of the raw intensities for the unmethylated (U) and methylated (M) channels, copy numbers (CN = M + U), Beta values (Beta) and M values (M-Val). The object contains also the raw control probes intensities and the results of the principal component analysis performed on the autosomal Beta values. The function also extracts the phenotype variables stored in the RGChannelSet. The summarization is done separately by probe types (I and II, see Bibikova et al.3) and for sex chromosomes. An S4 class, called shinyMethylSet, is used to represent the data in R, and this object is independent of the operating system. The shinyMethyl interface is launched by passing the shinyMethylSet to the function runShinyMethyl. An example of the interface is shown in Figure 2.\n\nThe interface shows an example interactive visualization of batch effects and quality control (TCGA head and neck squamous cell carcinoma, HNSCC dataset). The interface is divided into a user menu and a plotting area. (a) A menu containing a number of user-settable visualization parameters. The “phenotype” is set to “plate” which makes the color scheme reflect batch. The four plots (b–e) are interactive and react simultaneously to the user mouse clicks, so that samples selected on one plot are immediately highlighted on the additional plots. The solid lines in black represents the sample(s) currently selected by the user and match the dot circled in black on (b,c). The dashed lines in black represents another sample, previously selected by the user and match the black dot without the circle. (b) Average negative control probes intensities; (c) the median intensity of the M channel against the median intensity of the U channel; (d–e) M-value densities for Infinium I probes before and after functional normalization.\n\nSummarizing pre-processed data in shinyMethyl operates on an S4 object in minfi termed GenomicRatioSet. The summaries of the pre-process data are stored in an additional shinyMethylSet. Again, the summarized data object is substantially smaller than the full data representation in minfi. If this shinyMethylSet is also included in the runShinyMethyl command, the summaries of the pre-processed data are automatically added to the shinyMethyl interface. This option represents a powerful diagnostic tool to assess the global performance of a normalization method, such as plate effect correction (Figure 2), or preservation of the expected biological differences between different tissues or conditions (Figure 3).\n\nIn the first two plots are shown the densities of the M-values for Type I green probes before (a) and after (b) functional normalization as presented in the shinyMethyl interactive interface. Green and blue densities represent tumor and normal samples respectively, and red densities represent 9 technical replicates of a control cell line. The last two plots show the average density for each sample group before and after normalization. Functional normalization preserves the expected marginal differences between normal and cancer, while reducing the variation between the technical controls (red lines).\n\nOnce the DNA methylation data have been summarized, shinyMethyl offers three interactive plots for quality control. These plots react conjointly to the user mouse: (1) a density plot of the M/Beta values, (2) a QC plot proposed in minfi and (3) a plot of control probes intensities. The samples are colored by a phenotype variable selected by the user. The three plots together allow the user to select aberrant samples, whose array identifiers are saved into a csv file for exclusion in subsequent analyses (outside of shinyMethyl). An example of quality control panel is presented in Figure 2 in which summaries from the TCGA head and neck squamous cell carcinoma (HNSCC) samples are colored by batch; shinyMethyl allows to observe significant batch effects, a source of obscure variation that has critical consequences in downstream analysis6.\n\nThe sex of the samples can be accurately predicted by using the intensities of the probes mapping to the sex chromosomes in the M and U channels4. shinyMethyl implements this prediction algorithm and allows the user to interactively specify a cutoff to cluster samples by sex.\n\nThe array identifiers of the samples for which the predicted sex does not agree with the user-provided sex phenotype are displayed within the interface and can be saved into a csv file for further analysis. From the HNSCC TCGA dataset (described in Example data), one sample shows discrepancy, indicating possible mislabeling (Figure 4).\n\nThe difference of the median copy number intensity for the Y chromosome and the median copy number intensity for the X chromosome can be used to separate males and females. In a), the user can select the vertical cutoff (dashed line) manually with the mouse to separate the two clusters (orange for females, blue for males). Corresponding Beta-value densities appear in b) for further validation. The predicted sex can be downloaded in a csv file in c), and samples for which the predicted sex differs from the sex provided in the phenotype will appear in d).\n\nshinyMethyl also performs a principal component analysis (PCA) on the 20,000 most variable autosomal probes. This analysis enables the observation of associations between phenotype and methylation levels. An additional panel displays the physical arrays colored by phenotype. This coloring allows the user to discern potential confounding between phenotype and study design.\n\nThe data package shinyMethylData contains the summarized data for 369 HNSCC cancer samples from TCGA. It is available from the Bioconductor project (http://www.bioconductor.org). All analyses were performed on raw IDAT intensity files available from Level I data in the TCGA Data Portal (https://tcga-data.nci.nih.gov/tcga). Both raw intensities and normalized methylation values obtained by functional normalization using control probes and a slide covariate7 are included. The shinyMethylSet objects containing respectively the raw and normalized data can be accessed by summary.tcga.raw and summmary.tcga.norm.\n\n\nDiscussion\n\nshinyMethyl makes the quality control and pre-processing of 450k methylation array data fast and intuitive through an interactive application in R. We also show, by example, how to use shiny to develop interactive visualization interfaces. Our example will facilitate future developments of interactive visualization tools for the processing of high-dimensional genomic data in subsequent Bioconductor8 packages.\n\n\nSoftware availability\n\nshinyMethyl is an R package available from the Bioconductor project (http://www.bioconductor.org).\n\nhttps://github.com/jfortin1/shinyMethyl\n\nhttps://github.com/F1000Research/shinyMethyl/releases/tag/v1.0\n\nhttp://dx.doi.org/10.5281/zenodo.1074810\n\nArtistic-2.0", "appendix": "Author contributions\n\n\n\nJFP conceived and developed the shinyMethyl package, supervised by EJF and KDH. All authors wrote and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nJPF was partially supported by the Natural Sciences and Engineering Research Council of Canada and by les Fonds de recherche Nature et technologies du Québec as well as under the Johns Hopkins Head and Neck Cancer SPORE awarded to EJF.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nR Studio and Inc. shiny: Web Application Framework for R. R package version 0.10.0. 2014. Reference Source\n\nFeinberg AP, Vogelstein B: Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature. 1983; 301(5895): 89–92. PubMed Abstract \| Publisher Full Text\n\nBibikova M, Barnes B, Tsan C, et al.: High density DNA methylation array with single CpG site resolution. Genomics. 2011; 98(4): 288–95. PubMed Abstract \| Publisher Full Text\n\nAryee MJ, Jaffe AE, Corrada-Bravo H, et al.: Minfi: A flexible and comprehensive Bioconductor package for the analysis of Infinium DNA Methylation microarrays. Bioinformatics. 2014; 30(10): 1363–1369. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith ML, Baggerly KA, Bengtsson H, et al.: illuminaio: An open source IDAT parsing tool for Illumina microarrays. F1000Res. 2013; 2: 264. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeek JT, Scharpf RB, Bravo HC, et al.: Tackling the widespread and critical impact of batch effects in high-throughput data. Nat Rev Genet 2010; 11(10): 733–739. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFortin JP, Labbe A, Lemire M, et al.: Functional normalization of 450k methylation array data improves replication in large cancer studies. bioRxiv. 2014. Publisher Full Text\n\nGentleman RC, Carey VJ, Bates DM, et al.: Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 2004; 5(10): R80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFeinberg AP, Vogelstein B: Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature. 1983; 301(5895): 89–92. PubMed Abstract \| Publisher Full Text\n\nFortin JP, Hansen KD: F1000Research/shinyMhethyl. ZENODO. 2014. Data Source" }	[ { "id": "5619", "date": "19 Aug 2014", "name": "Timothy J. Triche", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title of the report is entirely appropriate. The abstract would benefit from mention of the interactive demo on spark.rstudio.com, as the interface is trivially easy to grasp while performing the various quality control checks. The methods not directly treated in this report (functional normalization, Shiny internals, 450k chip design) have been discussed elsewhere at length.The discussion of design and implementation decisions for shinyMethyl is sufficient (again an interactive/exploratory session will provide the interested user with most if not all of the same information). The conclusion (shinyMethyl eases 450k quality control for large datasets with potential technical artifacts) is justified in the report, the included example for the package, and the interactive demo hosted by Rstudio.The data for the example are available as raw binary IDAT files from the TCGA data portal; reproducibility via minfi is trivial, provided the user has sufficient compute resources to process and normalize a large dataset. (In my experience, datasets of 1000+ samples can be read with illuminaio and normalized via funnorm() on a typical Linux server with 48-64GB of RAM) This report serves a useful purpose (beyond the announcement of the software) by providing a usable baseline for 450k quality control. Epigenome-wide association studies are prone to a staggering number of potential confounding factors, not least of which are technical and experimental design artifacts. Investigators who have an interest in reproducible conduct of such studies now have further incentive to use and deposit raw binary IDAT files for their experiments. Investigators who choose not to do so may be viewed with skepticism, especially when the data has been processed for analysis via minfi (or, for that matter, methylumi), and verification of straightforward quality controls is made simpler by the interactive shinyMethyl package.", "responses": [ { "c_id": "982", "date": "15 Sep 2014", "name": "Kasper Daniel Hansen", "role": "Author Response", "response": "We thank the referee for taking the time to review our work, and thank for the kind review.We have submitted a revision with an added sentence about the demo at spark.rstudio.com under \"Software access\", but caution that this website at times is slow to respond to input. This is a hosting service freely available to the community." } ] }, { "id": "5620", "date": "03 Sep 2014", "name": "Tiffany J. Morris", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript written by Fortin et al. presents an R package to assess the quality control of 450k methylation array data through an interactive interface. A unique aspect of shinyMethyl is the use of the shiny package to allow the interactive visualisation. shinyMethyl is easy to use and therefore suitable for a novice data analyst with limited programming experience and also experienced bioinformaticians that would like to quickly assess large datasets. Additionally the package utilises the established and widely used R package minfi for the data analysis. shinyMethyl is available for download from Bioconductor or github. In this manuscript, the authors clearly describe the use of the package through a workflow and various screenshots and plots. They discuss the example dataset that is available through TCGA. Data interpretation is only explained briefly as the purpose of this manuscript is to present the interface. In depth background information on analysis methods (terminology, normalisations etc) and details of data interpretation can be found in the minfi publication or the Bioconductor vignette. The package does require the raw IDAT files. Some publicly available datasets may not provide this information and therefore shinyMethyl would not be applicable. However, it is becoming mainstream to provide this data and with more packages utilising this raw data it is expected that investigators make it available. The authors have clearly presented their package that is well designed, easy to use and a useful addition to the options for 450k data analysis especially as it is the platform of choice for large epigenome-wide association studies.", "responses": [ { "c_id": "981", "date": "15 Sep 2014", "name": "Kasper Daniel Hansen", "role": "Author Response", "response": "Thank you very much for taking the time to review our work and for the kind referee report." } ] } ]	1	https://f1000research.com/articles/3-175
https://f1000research.com/articles/2-210/v1	09 Oct 13	{ "type": "Research Article", "title": "Density but not climate affects the population growth rate of guanacos (Lama guanicoe) (Artiodactyla, Camelidae)", "authors": [ "María Zubillaga", "Oscar Skewes", "Nicolás Soto", "Jorge E Rabinovich", "Oscar Skewes", "Nicolás Soto", "Jorge E Rabinovich" ], "abstract": "We analyzed the effects of population density and climatic variables on the rate of population growth in the guanaco (Lama guanicoe), a wild camelid species in South America. We used a time series of 36 years (1977-2012) of population sampling in Tierra del Fuego, Chile. Individuals were grouped in three age-classes: newborns, juveniles, and adults; for each year a population transition matrix was constructed, and the population growth rate (λ) was estimated for each year as the matrix highest positive eigenvalue. We applied a stepwise regression analysis with population growth rate (λ) as dependent variable, and total guanaco population (in natural logs), annual mean precipitation, and winter mean temperature as independent variables, with and without time lags. The effect of population size was statistically significant, but the effect of the climatic variables on guanaco population growth rate was not significant.", "keywords": [ "Camelidae", "climatic variables", "density effects", "guanaco", "population regulation", "population growth rate" ], "content": "Introduction\n\nIn order to understand population dynamics and optimize the management of wildlife populations it is important to identify how groups are affected by environmental factors and their own density, particularly in species living in extreme environments. Mechanisms that regulate populations of the guanacos (Lama guanicoe) are poorly known; this species is heavily exploited in the Patagonian steppe, and its numbers and distribution have diminished significantly during the last century because of grazing conflicts with a sheep-based society, and overhunting (the guanaco’s historical distribution has been reduced by 75% in Chile and Peru, and by 60% in Argentina)1. The guanaco is in the Least Concern IUCN Red List category (http://www.iucnredlist.org/, downloaded on 11 September 2013) but it has been included in Appendix II of CITES 2013 (http://www.cites.org/eng/app/2013/E-Appendices-2013-06-12.pdf).\n\nNo population of any species can grow indefinitely, and population checks based upon different processes restrict population size and/or geographic distribution; these processes are either density-stabilizing or density-limiting2, the latter being independent of population size. Stabilization results from density-dependence, with a regulatory effect that varies in intensity with the size or density of the population itself. However, density-dependent processes are also affected by environmental conditions, and many wildlife population dynamic and management models include the effects of climatic covariates (e.g., Dennis & Otten, 2000; Colchero et al., 2009)3,4. In the case of the Ricker model (one of the most simple and most used population models), Corani and Gatto (2007)5 proposed an original way of incorporating climatic covariates: they were included as affecting the population growth rate.\n\nThe guanaco is one of the two extant wild South American camelids, and ranges from Northern Peru through Chile, and across Patagonia to southern Argentina and Chile, reaching Tierra del Fuego. It is found from sea level to nearly 4500 m on the Andes mountain range, and occupies a wide variety of habitats from hardpan deserts to scrublands to grasslands6.\n\nAlthough there are several studies on the population regulation processes in mammals in general7,8, and in ungulates in particular9–12, there are very few studies in wild South American camelids that analyze population growth regulation. There have been some studies on populations of vicuña (Vicugna vicugna)13–15 and some on populations of guanacos16–18; however none of these studies considered the direct effect of density and/or climate on population growth rate.\n\nThus, we tested the hypothesis that population density and environmental covariates, such as average winter temperature and average annual precipitation, affect the growth rate of the guanaco population from the island of Tierra del Fuego, Chile.\n\n\nMaterials and methods\n\nWe analyzed the guanaco population of the “Cameron” ranch (-53.9 S, -69.3 W), with an area of the 2000 km2 located in the Southern region of the Tierra del Fuego island, Chile. The altitude range is 0–300 m above sea level, and the ranch is a mosaic of steppe and forest biomes; the latter is a deciduous forest and the steppe is composed of meadows, peats and prairies. The guanaco is the dominant herbivore, with the exception of sheep (the dominant domestic species) with densities that have fluctuated in the last decades between 11 and 23 sheep/km226. Based on metabolism studies19, the guanaco/sheep equivalence factor is 1.65 (i.e., each sheep is equivalent to 0.61 guanacos), close to the values of 1.5 and 1.8 sheep per guanaco proposed by Raedeke (1978)20 based on diet habits. In non-forested areas (the Patagonian steppe) the climate is characterized by an average annual precipitation of 200–400 mm, while in the forested areas the average precipitation fluctuates between 400–600 mm per year20. The average annual temperature is 6.5°C and the average winter temperature is 2.2°C.\n\nGuanacos were counted for 34 consecutive years between 1977 and 2012 (except 1986 and 1996), using the transect method with a variable width, and a maximum of 1000 m to each side of the transect from 1977 to 2000, and with a fixed width band from 2001 to 2012 (the latter with a maximum of 500 m to each side of the transect)21–25. The sampling period was carried out in the autumn and lasted approximately 7 days between 10:30 and 19:00 h, with two observers in two 4x4 vehicles going over the main, secondary and local roads at a maximum speed of 40 km/h. Each road was covered only once, and in addition to individual guanaco counts, the following were recorded: weather conditions, time, distance (km) from the starting point, GPS coordinates, observation distance from the transect (m), an estimate of the angle to the animal’s position, and – when the animals were observed in groups – the number of individuals, the type of social group, and its structure (sex, and the age class: newborn, juvenile and adult). Observations were done with model Trophy of Bushnell® 10x42 binoculars, and the GPS was a Garmin model eTrex Vista. Observation distances were made by naked eye, but the four observers had been trained in this technique using laser telemeters, GPS and the vehicles’ odometers to calibrate the estimated distances. The road network and all geo-referenced observations were processed with the Arc View 9.3 Geographical Information System (SIG), and transferred using program Map Source®. The cartography was kindly provided by the Chilean “Servicio Agrícola y Ganadero” (SAG).\n\nThe population size was estimated as given in Soto26 which was based in the method described by Raedeke (1978)20, given by:\n\n\n\nwhere N is the total population to be estimated, A is the total study area, n is the total number of animals counted, x is the total transect distance covered (m) rounded to one meter, and y is the average of the perpendicular distance (m) from the transect to the animals counted (the factor of 2 is included to consider that there is one band to each side of the transect). The variance (S2) was estimated by (2), with p = n/N, and used to estimate the 95% upper and lower confidence intervals.\n\n\n\nIn addition to the direct density-dependence, we evaluated the possible impact of two environmental covariates on guanaco population dynamics: average annual precipitation and winter temperature (as the average of months of June, July and August). We used the 25 years (1977–2002) precipitation and temperature time series of the CRU TS 2.1 database, compiled by the Tyndall Centre, Climatic Research Unit, School of Environmental Sciences of the University of East Anglia, United Kingdom (http://www.cru.uea.ac.uk/cru/data/hrg.htm). As the CRU TS 2.1 data ended in 2002, we completed that time series for 2003 to 2012 from the closest meteorological station to the Cameron ranch: Punta Arenas (Chile); this data was downloaded from the Internet site of the Meteorological Service of Chile (http://www.meteochile.gob.cl/).\n\nWe estimated the population growth rate (as measured by the finite net rate of increase, λ) for each of the 36 years of the guanaco data (though population values were not available for years 1986 and 1996, and were linearly interpolated) by fitting a three age-class (newborns, juveniles and adults) matrix model to the field population estimates; as the transition matrix is a female only matrix we summed the three age classes and multiplied by two (in this population there is a 50% sex-ratio) to carry out the fit with the total field population. The fit was carried out in an Excel® spreadsheet using the Solver tool, and the sum of squares (SSQ) goodness of fit criterion was used (details can be found in Rabinovich and Zubillaga, 201227); this process resulted in a set of 36 population stage-structured matrices. From each matrix we calculated the largest positive eigenvalue28 as an estimate of λ; with the PopTools add-in, an Excel program developed by Greg Hood (http://www.cse.csiro.au/poptools/).\n\nWe checked the relationship between the population growth rate (as measured by the finite net rate of increase, λ) and population size (in units of natural logs) and/or climate using a stepwise regression analysis with λ as dependent variable, and total guanaco population (in natural logs), annual mean precipitation (mm/year), and winter mean temperature (°C) as independent variables. For the climatic covariates we also evaluated the effect of time lags (T), with T = 1 to 7 years for precipitation, and T = 1 year for winter temperature; the lags were applied by averaging the previous T lagged years, as suggested by Shaw et al. (2012)14. These regressions were carried out using the statistical package Statistica (StatSoft, 2009; http://www.statsoft.com/). Since it is necessary to corroborate that there is no collinearity (significant correlations among the predictor variables) for multiple regressions analysis, we carried out correlation analysis between independent variables before the stepwise analysis. A p value < 0.05 was considered significant.\n\n\nResults\n\nThe table in Supplementary Table 1 shows the guanaco abundance data for the 36 years of sampling, the estimated values of the population rate of growth (λ) from the 36 population transition matrices, and the values of climatic variables used in the regression analysis.\n\nThe precipitation covariates with 1 to 7 year lags showed a statistically significant Pearson's correlation coefficient (p < 0.05), while the other independent variables didn’t show a statistically significant correlation. Thus, for the stepwise analyses we chose one of these correlated covariates (precipitation with lag T = 4). The results indicated that the total population (LnNtot) was the only statistically significant variable (Table 1), and the model was statistically significant (p < 0.030 and F(5, 30) = 2.885). Table 1 shows both the standardized and the raw regression coefficients, with their corresponding standard errors. The magnitude of these standardized regression coefficients allows comparison of the relative contribution of each independent variable in the prediction of the dependent variable.\n\nThe results show the standardized regression coefficients (Sz. Reg. coeff.), their standard errors (Std.E. SRC), the raw regression coefficients (Reg. Coeff.), their standard errors (Std.E. RC) and t-test values (t-stud.).\n\nWe carried out another regression analysis using λ as the dependent variable and only population size (in natural logs, LnNtot) as an independent variable, in order to compare it with the full model (population size and climatic covariates as independent variables). Table 2 shows the result of this second regression analysis, which was highly significant (p < 0.0018, r = 0.502); the regression equation was λ = 1.6373–0.0552 (±0.0163) LnNtot; a comparison of both regressions using a two-way ANOVA test indicated that there was no statistical significant differences between them (F = 0.803, p = 0.5329).\n\nInterpretation of the statistical parameters as in Table 1. The results show the standardized regression coefficients (Sz. Reg. coeff.), their standard errors (Std.E. SRC), the raw regression coefficients (Reg. Coeff.), their standard errors (Std.E. RC), and t-test values (t-stud.).\n\nThe negative slope is an indication of a density-dependent process, since when the population increases the per capita population growth rate decreases (Figure 1). The intercept of the regression line at λ = 1 (population at equilibrium) results in a population size of 103,000 guanacos (51 guanaco/km2), which can be considered as an estimate of the carrying capacity of the Cameron ranch for this guanaco population.\n\nOn the x-axis the guanaco population from the Cameron ranch (Tierra del Fuego, Chile) is given in a natural logarithmic scale. The values of λ represent the finite population growth rate.\n\n\nDiscussion\n\nWe were not able to confirm the expected effect of climatic covariates on the population rate of growth in the guanaco population of the Cameron ranch. We expected such an effect because Sarno et al. (1999)18 found a relationship between winter weather and guanaco yearling’s survival, and Rey et al. (2012)29 recorded an effect of drought on guanaco fecundity. Thus we anticipated that as winter temperature and/or annual precipitation decrease, the population growth rate should also decrease. Our negative results are even more surprising because in the last 10–12 years the guanaco population fluctuated remarkably, suggesting some sort of interaction between population density and climatic covariates. It is well known that environmental effects are more important when the population size is near the carrying capacity, particularly in large mammals (as the guanaco) characterized by low reproductive rates, long life-spans, and populations that are resource-limited (features typical of species referred to as the “K-selected” species)30. The difference between our results and that of Sarno et al. (1999)18 may reflect that these authors used the average winter snowfall while we considered average winter temperature.\n\nTaper & Gogan (2002)31 carried out a study with the Yellowstone elk similar to our analysis, although the weather variables were included as covariates within a dynamic model (exponential, Ricker and Gompertz models). They found that spring precipitation had a positive regression coefficient, a result opposite to our conclusion for the guanaco population.\n\nOur conclusion is that in order to test the effects of climatic covariates on population regulation of large ungulates, the use of a population dynamic model is recommended, for they may be more sensitive to the interaction between density-dependent processes and weather variables, than a simple regression between them and population growth rate. For example, our negative results do not conform to what was obtained by Shaw et al. (2012)14 with respect to a very phylogenetically related camelid species: the vicuña. Using a logistic model fitted to 31 years of data these authors found that rainfall had a highly significant effect on population size with a time lag of 4 years, while in our guanaco regression analysis, no climatic covariate was statistically significant.\n\nWe conclude that, opposite to what we expected based on the bibliography of ungulate’s population dynamics, weather variables do not seem to influence the density-dependent population growth rate process.", "appendix": "Author contributions\n\n\n\nMZ and JER conceived the study. NSV and OSR sampled and estimated the population size. MZ designed the analysis. MZ and JER were responsible for all writing phases of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to publish the article.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nSupplementary table\n\nPopulation values were not available for years 1986 and 1996, and were linearly interpolated.\n\n\nReferences\n\nFranklin WL, Bas MF, Bonacic CF, et al.: Striving to manage Patagonia guanacos for sustained use in the grazing agroecosystems of southern Chile. Wildl Soc Bull. 1997; 25(1): 65–73. Reference Source\n\nCase TJ: An illustrated guide to Theoretical Ecology. Oxford, University Press; 2000; 449. Reference Source\n\nDennis B, Otten M: Joint effects of density dependence and rainfall on abundance of san joaquin kit fox. J Wildl Manage. 2000; 64(2): 388–400. Reference Source\n\nColchero F, Medellin RA, Clark JS, et al.: Predicting population survival under future climate change: density dependence, drought and extraction in an insular bighorn sheep. J Anim Ecol. 2009; 78(3): 666–673. PubMed Abstract \| Publisher Full Text\n\nCorani G, Gatto M: Structural risk minimization: a robust method for density-dependence detection and model selection. Ecography. 2007; 30(3): 400–416. Publisher Full Text\n\nFranklin WL, Johnson WE: Hand Capture of Newborn Open-Habitat Ungulates: The South American Guanaco. Wildl Soc Bull. 1994; 22(2): 253–259. Reference Source\n\nOstfeld RS, Canham CD, Pugh SR: Intrinsic Density-Dependent Regulation of Vole populations. Nature. 1993; 366(6452): 259–261. PubMed Abstract \| Publisher Full Text\n\nBateman AW, Ozgul A, Coulson T, et al.: Density dependence in group dynamics of a highly social mongoose, Suricata suricatta. J Anim Ecol. 2012; 81(3): 628–639. PubMed Abstract \| Publisher Full Text\n\nKoons DN, Terletzky P, Adler PB, et al.: Climate and density-dependent drivers of recruitment in plains bison. J Mammal. 2012; 93(2): 475–481. Publisher Full Text\n\nBardsen BJ, Tveraa T: Density-dependence vs. density-independence - linking reproductive allocation to population abundance and vegetation greenness. J Anim Ecol. 2012; 81(2): 364–376. PubMed Abstract \| Publisher Full Text\n\nSerrano E, Angibault JM, Cargnelutti B, et al.: Density dependence of developmental instability in a dimorphic ungulate. Biol Lett. 2008; 4(5): 512–514. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStewart KM, Bowyer RT, Ruess RW, et al.: Herbivore Optimization by North American Elk: Consequences for Theory and Management. Wildl Monogr. 2006; 167(1): 1–24. Publisher Full Text\n\nRabinovich JE, Hernández MJ, Cajal JL: A simulation model for the management of vicuña populations. Ecol Model. 1985; 30(3–4): 275–295. Publisher Full Text\n\nShaw AK, Galaz JL, Marquet PA: Population dynamics of the vicuña (Vicugna vicugna): density-dependence, rainfall, and spatial distribution. J Mammal. 2012; 93(3): 658–666. Publisher Full Text\n\nBonacic C, Macdonald DW, Galaz J, et al.: Density Dependence in the camelid Vicugna vicugna: the Recovery of a Protected Population in Chile. Oryx. 2002; 36(2): 118–125. Publisher Full Text\n\nSarno RJ, Franklin WL: Population Density and Annual Variation in Birth Mass of Guanacos in Southern Chile. J Mammal. 1999; 80(4): 1158–1162. Reference Source\n\nSarno RJ, Franklin WL: Maternal expenditure in the polygynous and monomorphic guanaco: suckling behavior, reproductive effort, yearly variation, and influence on juvenile survival. Behav Ecol. 1999; 10(1): 41–47. Publisher Full Text\n\nSarno RJ, Clark WR, Bank MS, et al.: Juvenile guanaco survival: management and conservation implications. J Appl Ecol. 1999; 36(6): 937–945. Publisher Full Text\n\nWarmington BG, Wilson GF, Barry TN: Voluntary Intake and Digestion of Ryegrass Straw by Llama X Guanaco Crossbreds and Sheep. J Agric Sci. 1989; 113(1): 87–91. Publisher Full Text\n\nRaedeke KJ: El guanaco de Magallanes, Chile. Su distribución y Biología. Corporación Nacional Forestal Publicación Técnica N° 4, Ministerio de Agricultura Chile; 1978; 364. Reference Source\n\nTellería Jorge JL: Manual para el censo de los Vertebrados terrestres. Editorial Raíces., Madrid, España; 1986; 278. Reference Source\n\nDavis D, Winstead R: Estimación de tamaños de poblaciones de vida silvestre. In: H.S. Mosby, R.H. Giles Jr. y S.D. Schemnitz (Eds). Manual de técnicas de gestión de vida silvestre. The Widlife Society, Bethesda, Maryland; 1987; 233–258.\n\nCaughley G: Analysis of vertebrate populations. A Wiley-Interscience publication. John Wiley & Sons Ltd; 1980; 234. Reference Source\n\nBuckland ST, Anderson DR, Burnham KP, et al.: Distance Sampling: Estimating Abundance of Biological Populations. Chapman and Hall, London; 1993; 446. Reference Source\n\nBuckland ST, Anderson DR, Burnham KP, et al.: Introduction to Distance Sampling. Oxford, University Press; 2001. Reference Source\n\nSoto Volkart N: Distribución y Abundancia de la Población de Guanacos (Lama guanicoe, Muller 1776) en el Área Agropecuaria de Tierra del Fuego (Chile) y su Relación de Carga Animal con la Ganadería Ovina. Tesis de Estudios Avanzados (Dea). Universidad Internacional de Andalucía - Universidad de Córdoba; 2010.\n\nRabinovich JE, Zubillaga M: Informe Final del proyecto: \"Modelo de manejo de poblaciones de guanacos para la Provincia del Chubut\".2012. Reference Source\n\nCaswell H: Matrix population models: construction, analysis, and interpretation. 2nd edn. Sinauer Associates. Sunderland, Massachusetts; 2001; 722. Reference Source\n\nRey A, Novaro A, Sahores M, et al.: Demographic effects of live shearing on a guanaco population. Small Ruminant Research. 2012; 107(2): 92–100. Publisher Full Text\n\nFowler CW: Density Dependence as Related to Life History Strategy. Ecology. 1981; 62(3): 602–610. Publisher Full Text\n\nTaper ML, Gogan PJP: The Northern Yellowstone Elk: Density Dependence and Climatic Conditions. J Wildl Manage. 2002; 66(1): 106–122. Reference Source" }	[ { "id": "2048", "date": "20 Nov 2013", "name": "Ricardo Baldi", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors hypothesized that population density and climatic variables affect the growth rate of the population of guanacos in the study area. However, as it is detailed under the “Study area” subsection, the domestic sheep share the area with the guanaco and substantial changes in stocking rate have been reported for the ranch during the study period. It is known that sheep and guanacos select similar diets across different parts of the range they share, and usually sheep presence and abundance explain a substantial amount of variation in the abundance of guanacos, included the study area comprising the Estancia Cameron as described by Raedeke (1979). In his work, Raedeke described that sheep are managed “rotationally” around the property, taken seasonally to different areas in winter and summer, and this makes guanacos move seasonally to the forest habitat. Therefore, it would be expected that variations in sheep abundance, not only seasonally but in total numbers through time, could affect the estimates of guanaco abundance. In my opinion, this is a major issue in the design of this work. Also, as explained below, the survey design of this work could play a key role in the quality of the estimates obtained, which is reflected also in the interpretation of results. All of these do not mean that density dependent processes are not likely to occur, but to my understanding it is not possible to rule out other processes to be conclusive.Guanaco population samplingThe surveys were conducted at a 2,000 km2 ranch where the habitat is a mosaic of steppe and forest biomes. The authors indicate that they surveyed guanacos using the “transect method with a variable width” from 1977 to 2000. Is this the distance sampling? If so, it is based on measuring the perpendicular distance from the transect to the object. Also, it is says that the surveying methodology was changed to “fixed width band” in 2001 and subsequent years. Some comments and questions on these issues regarding survey design are:Were both the steppe and forest habitats surveyed using the same methodology? Transect lines to observe individuals and make direct counts are commonly used in open habitats, but not in the forest. However if this was not the case, the authors need to account for the variation in the probability of detection as it is expected to vary significantly between such contrasting habitat types. There was a change in the surveying method, which also brings the point of detection probability. While the line transect method assumes that the probability of detecting objects on the line (distance=0) is p=1, and as objects are far from the line the probability of detection follows a certain declining function, the main assumption of the strip transect method is that the probability of detecting objects within the strip is 1 (i.e. no objects are left undetected). This variation in the probability of detection according to the methodology applied during each period must be discussed. Also, the ways that the density estimates were obtained are unclear and must be specified (i.e. distance sampling, other calculation methods). Regarding the estimate of population size, several considerations can be made about the extrapolation of absolute densities to population numbers. First, the spatial pattern of the transects surveyed could be determinant of biased estimates, if the objects are not randomly distributed in relation to the survey line (i.e. if the transect follows a geographic feature in such a way the animals are either attracted or deterred). Perhaps the transects are located across certain areas – for example the steppe – but not the forest and thus the estimate could be biased towards the density calculated for the surveyed portion of the total area. This is especially important at the time of extrapolate densities to obtain population size numbers, as densities are strictly valid for the areas effectively surveyed while traveling along the transect lines. In any case, it would be useful to provide the distribution of surveyed lines across the area. The second point I find relevant to discuss regarding survey design is the proportion of the area effectively surveyed. How many km2 were comprised by the lines and strips surveyed? Again, the extrapolation of densities to population size at a larger scale must account for possible limitations related to the survey design and effort invested.Results and discussionMajor fluctuations of the guanaco population occurred during the last 10-12 years analyzed, as can be found in the additional data file provided, and also pointed out by the authors. But this variation could well be due to the changes in methodology at the time of the population surveys. As said above, it is necessary to provide detailed information on the type of survey conducted, the differences with the previous method and the way the density estimates were obtained. It would be informative if the authors provide the errors associated with the population estimates.As proposed above, sheep densities could be affecting guanaco population trends, but the design of the work does not allow accounting for this factor. In my opinion, sheep densities must be included as an explanatory variable as there is evidence they affect guanaco numbers. Also, as the authors concluded that in this study the guanaco populations was near carrying capacity, and this must be common to both guanacos and sheep, thus sheep must be considered in the analysis as its density was markedly variable during the study period. Another factor that should be considered at the time of the discussion is the possibility of guanacos moving from the surveyed surroundings or even from Cameron to neighboring sites, as they may respond to changes in the spatial availability of resources by occupying different sites. In summary, the marked fluctuations in population size require the examination and discussion of other possible factors before concluding that density dependent processes would drive population trends.", "responses": [ { "c_id": "823", "date": "14 May 2014", "name": "Maria Zubillaga", "role": "Author Response", "response": "\"The authors hypothesized that population density and climatic variables affect the growth rate of the population of guanacos in the study area. However, as it is detailed under the “Study area” subsection, the domestic sheep share the area with the guanaco and substantial changes in stocking rate have been reported for the ranch during the study period. It is known that sheep and guanacos select similar diets across different parts of the range they share, and usually sheep presence and abundance explain a substantial amount of variation in the abundance of guanacos, included the study area comprising the Estancia Cameron as described by Raedeke (1979). In his work, Raedeke described that sheep are managed “rotationally” around the property, taken seasonally to different areas in winter and summer, and this makes guanacos move seasonally to the forest habitat. Therefore, it would be expected that variations in sheep abundance, not only seasonally but in total numbers through time, could affect the estimates of guanaco abundance. In my opinion, this is a major issue in the design of this work. Also, as explained below, the survey design of this work could play a key role in the quality of the estimates obtained, which is reflected also in the interpretation of results. All of these do not mean that density dependent processes are not likely to occur, but to my understanding it is not possible to rule out other processes to be conclusive.\" In relation to the suggestion to incorporate livestock in the analysis, we re-analyzed our regression incorporating the variable livestock as sheep population; we re-processed the regressions incorporating the sheep population as a new independent variable of the guanaco population growth rate (l); so our new analysis increased from 5 to 6 independent variables: sheep population, guanaco population size, winter temperature, winter temperature with a lag of 1 year, annual precipitation, annual precipitation with a lag of 3 years. Our main results didn’t change and it was confirmed that only the guanaco population size seems to have an effect on the population growth rate (l). These changes can be found in the “Methods” and in the “Results” paragraphs. \"The surveys were conducted at a 2,000 km2 ranch where the habitat is a mosaic of steppe and forest biomes. The authors indicate that they surveyed guanacos using the “transect method with a variable width” from 1977 to 2000. Is this the distance sampling? If so, it is based on measuring the perpendicular distance from the transect to the object.\"No, the sampling methodology did not use the program Distance. The citations of Buckland et al. (1993 and 2001) were removed to avoid possible misinterpretations with the sampling methodology used.The method used to estimate the population size was the King method modified by Leopold (1933) as described in Raedeke (1978). In the manuscript it was explained and justified this method with the following paragraph:“Raedeke (1978) 19 claims that the Leopold method is valid when the following conditions are satisfied: (1) the road systems must be an adequate sampling of the study area, (2) the network road must be randomly distributed, and (3) the animals included in the sampling must be randomly distributed in relation to the observer’s route; and he considers that in the south of the Tierra del Fuego Island these assumptions are fulfilled. Soto 201018 compared population estimations by the Leopold and Distance methods and found that the value of the means estimate by Leopold methods fall within the confidence intervals estimated by Distance; additionally, as all sampling periods used the same methodology whatever bias might exist in the estimation of the mean abundance using the Leopold method, the relative changes among years (and thus the temporal trend) will be adequately represented; thus, we conclude that the Leopold’s method seems adequate for our purposes18. The area effectively surveyed in each sampling period was around 420 km² (about 20%, of the area under study).”\"Also, it is says that the surveying methodology was changed to “fixed width band” in 2001 and subsequent years. Some comments and questions on these issues regarding survey design are:Were both the steppe and forest habitats surveyed using the same methodology? Transect lines to observe individuals and make direct counts are commonly used in open habitats, but not in the forest. However if this was not the case, the authors need to account for the variation in the probability of detection as it is expected to vary significantly between such contrasting habitat types.\"We corrected in the manuscript the years where the change in band width occurred: variable band width from 1977 to 1995, and with a fixed width band (with a maximum of 500 m to each side of the transect) from 1996 to 2012.The area occupied by forest is low and it has some degree of intervention, the visibility of the guanaco is adequate, hence we considered that the forest is a minor component and it does not affect the efficiency of sampling. In the section “Study area” the following paragraph was incorporated:\"The forested area is about 8.8% of the total study area, with different degrees of forest clearance that offers adequate visibility for guanaco sampling.” \"There was a change in the surveying method, which also brings the point of detection probability. While the line transect method assumes that the probability of detecting objects on the line (distance=0) is p=1, and as objects are far from the line the probability of detection follows a certain declining function, the main assumption of the strip transect method is that the probability of detecting objects within the strip is 1 (i.e. no objects are left undetected). This variation in the probability of detection according to the methodology applied during each period must be discussed. Also, the ways that the density estimates were obtained are unclear and must be specified (i.e. distance sampling, other calculation methods).\"We improved this section and the next paragraph was incorporate to explain this change. The population size was estimated by King method modified by Leopold (1933) as described by Raedeke (1978), which was also clarified in the manuscript.\"The sampling methodology was changed by an expert recommendation made in 1995 23; as in year 2000 the fixed and the undefined band width methods were applied simultaneously, we tested both results and found no significant statistical difference between them.” \"Regarding the estimate of population size, several considerations can be made about the extrapolation of absolute densities to population numbers. First, the spatial pattern of the transects surveyed could be determinant of biased estimates, if the objects are not randomly distributed in relation to the survey line (i.e. if the transect follows a geographic feature in such a way the animals are either attracted or deterred). Perhaps the transects are located across certain areas – for example the steppe – but not the forest and thus the estimate could be biased towards the density calculated for the surveyed portion of the total area. This is especially important at the time of extrapolate densities to obtain population size numbers, as densities are strictly valid for the areas effectively surveyed while traveling along the transect lines. In any case, it would be useful to provide the distribution of surveyed lines across the area. The second point I find relevant to discuss regarding survey design is the proportion of the area effectively surveyed. How many km2 were comprised by the lines and strips surveyed? Again, the extrapolation of densities to population size at a larger scale must account for possible limitations related to the survey design and effort invested.\"Although the transects are not randomly distributed, the preexisting roads can be considered as an adequate sample of the area. This comment was incorporate in the manuscript. \"Despite randomly selected transects are recommended24, preexisting roads were used because according to Soto (2010)18 the existing system of roads is an adequate sample of the area.”The second point was considered and the area effectively surveyed was estimated. In the “Guanaco population sampling” section the following paragraph was incorporated:\"The area effectively surveyed in each sampling period was around 420 km² (about 20%, of the area under study).” \"Major fluctuations of the guanaco population occurred during the last 10-12 years analyzed, as can be found in the additional data file provided, and also pointed out by the authors. But this variation could well be due to the changes in methodology at the time of the population surveys. As said above, it is necessary to provide detailed information on the type of survey conducted, the differences with the previous method and the way the density estimates were obtained. It would be informative if the authors provide the errors associated with the population estimates.\"The errors associated with the population estimates are given in Table S1 where the lower and upper limits of the 95% confidence intervals (CI) are shown. \"As proposed above, sheep densities could be affecting guanaco population trends, but the design of the work does not allow accounting for this factor. In my opinion, sheep densities must be included as an explanatory variable as there is evidence they affect guanaco numbers. Also, as the authors concluded that in this study the guanaco populations was near carrying capacity, and this must be common to both guanacos and sheep, thus sheep must be considered in the analysis as its density was markedly variable during the study period. Another factor that should be considered at the time of the discussion is the possibility of guanacos moving from the surveyed surroundings or even from Cameron to neighboring sites, as they may respond to changes in the spatial availability of resources by occupying different sites. In summary, the marked fluctuations in population size require the examination and discussion of other possible factors before concluding that density dependent processes would drive population trends.\"This factor (sheep population) has been incorporated, and the corresponding analysis was carried out; see in the “Materials and methods” section “Sheep Population” and the section “Results”. In the “Discussion” section the following paragraph was included where we recognized the possibility of guanaco moving as possible source of error in the sampling:“The results of the guanaco population sampling suggest a certain trend in the population size, with a sort of exponential growth in the first few years, becoming more variable as the population grows, with more marked fluctuations during the last 10-12 years; this may imply that the population is becoming stabilized, possibly approaching its carrying capacity. On the other hand, the sampling results of those last 10-12 years show abrupt “jumps” in some years (for example 2004-2005) that may be the consequence of the mobility of guanacos from the Cameron ranch to neighboring sites and vice versa; since the transect sampling does not identify individuals the effects of possible local displacements could not be considered.”" } ] }, { "id": "2847", "date": "19 Dec 2013", "name": "Pablo Acebes", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors explore the population dynamics of a long-term monitored guanaco population (36 years) in the Chilean Tierra del Fuego, trying to discern if the process is governed by population size and/or environmental (climatic) variables, assuming that guanaco growth rate follows a density-dependent process. Although the topic is very interesting, the authors have room to improve the manuscript. Below the authors will find major and minor comments.Major commentsThe manuscript is classified as a “Research article” when indeed it seems a “Short note”. It would greatly benefit from deeper and more detailed explanations of:The theory of ungulate population dynamics, driven by density-dependent and density-independent processes; please expand this topic in the introduction and discuss more deeply your results considering the theoretical framework (see comments below). The census methodology (see comments below). Statistical modeling (see comments below).Ungulate populations are regulated via endogenous forces (density-dependent processes), and exogenous forces (density-independent): weather and predation. Weather determines the primary production, i.e. the resource (forage) availability, which in turn determines the carrying capacity. On the other hand weather, through extreme conditions (e.g. severe winter climate), modulates population dynamics (starvation, survival). Predation is considered an important force which shapes population dynamics as well. In addition, poaching or overhunting act as predation, and competition with other ungulates is another important exogenous force. With this framework authors should justify:The background supporting the study, as it currently is a bit poor, in my view. The authors should explain more in detail, for example, which are the differences between the density-stabilizing and the density-limiting processes, or the evidence of including climatic variables when modelling population growth rate trends. The use of these climatic variables: the average annual precipitation and winter temperature. Without any justification, authors could have also included others variables in the models, as climatic variables do not account for the pattern described, but this doesn’t necessarily mean that climate hasn’t got any effect on the guanaco population growth rate. Moreover authors should explain if these two climatic variables are selected as indicators of primary production and harsh weather conditions. As authors stated in introduction, guanaco populations have undergone overhunting (poaching) and competition with sheep, these being two of the major forces of their dramatic decline in the last century. For that reason, it is quite surprising that no variables related to livestock were included in the modelling procedure. The population has increased a lot in the studied period (x8) and just climate and/or density-dependent process do not explain alone such increase in my view. Therefore authors should consider including livestock effects, its influence on resource availability along the studied period and the mentioned overhunting, as this could really explain the detected trend.The guanaco sampling method has changed during the studied period (different band widths). Besides, were the transects chosen based on the topography and vegetation cover of the ranch to ensure that all areas were visible to census-takers along their transects? I am not sure if that could be a source of error, but authors should discuss this point. Moreover, I recommend authors to re-analyze their data using the DISTANCE program (Buckland et al., 2004) to estimate population size over the period, as the analyses performed by this package seems much more robust that the one employed by authors. If they do so, authors could also include topographic variables in the models to see whether the resulting models change. The reason is that the authors do not explain if guanacos occur in both biomes (steppe and forest), because if that was true, detection of guanacos was completely different in both ecosystems. Therefore the band width of 500 or 1000m is not valid for forests.Regarding the population size estimated along the 36 years (Supplementary table), important (huge) differences are found, for example between 2004 and 2005 or between 2006 and 2007, or between 2007 and 2008. My concern is if such “jumps” could be related to the census surveyed. Please discuss this point.The analytical approach employed by the authors doesn’t seem appropriate in my view, as they also recognize in the discussion, and that could be the reason for not finding any climatic variable significant in the model:The authors use a stepwise regression when other approaches such as logistic regressions, Ricker or Beverton–Holt models could give more suitable results. Please explain or justify your decision. The selection of the best model among the candidates should be done by Akaike’s information. Minor commentsRefer to climatic variables or climatic effects, instead of climatic covariables. Explain more in detail Corani & Gatto’s studies. Please remove information related to metabolic studies between guanacos and sheep (1st paragraph of study area section). Please add Nothofagus spp among the brackets after deciduous forest (study area section) if it is the case. Give more information about livestock ranching, density, and size and the relation of livestock rangers with guanacos along the studied period (study area section). Are the guanacos found both in the steppe and the forest biomes? Please re-write the paragraph related to guanaco sampling through transect method in the consecutive periods (it is not clear enough). Please remove information related to binoculars and GPS brands (did the authors use the same gadgets across 36 years?). Explain the interest of using time lags (T) as covariables in the model. Please add the Statistica version employed. Please move explanations of the analyses perform from results to material & methods section.", "responses": [ { "c_id": "822", "date": "14 May 2014", "name": "Maria Zubillaga", "role": "Author Response", "response": "\"The background supporting the study, as it currently is a bit poor, in my view. The authors should explain more in detail, for example, which are the differences between the density-stabilizing and the density-limiting processes, or the evidence of including climatic variables when modelling population growth rate trends.\"After the following paragraph that was already in the original text:“No population of any species can grow indefinitely, and population checks based upon different processes restrict population size and/or geographic distribution; these processes are either density-stabilizing or density-limiting”We added the following paragraph explaining the differences between the density-stabilizing and the density-limiting processes.“The former are of a biotic nature and depends on the interaction between individuals of the same or different species, while the latter are independent of population size. Stabilization results from density-dependence, with a regulatory effect that varies in intensity with the size or density of the population itself, however, not all density-dependent factors are density-stabilizing. The density-limiting factors can also be considered density-responsive because the per capita amount or availability of resources decreases as the population density increases. Thus, these two types of factors (density-stabilizing and density-limiting) rarely act independently: the density-limiting factors (generally of a physical and/or climatic nature), may determine the level at which populations become stabilized by the density-dependent processes, but they do not have a stabilizing capacity per se; for this reason many wildlife population dynamic and management models include the effects of climatic variables (e.g., Dennis & Otten, 2000; Colchero et al., 2009)3,4”. \"The use of these climatic variables: the average annual precipitation and winter temperature. Without any justification, authors could have also included others variables in the models, as climatic variables do not account for the pattern described, but this doesn’t necessarily mean that climate hasn’t got any effect on the guanaco population growth rate. Moreover authors should explain if these two climatic variables are selected as indicators of primary production and harsh weather conditions.\"To explain and justify the use of these climatic variables the paragraph below was incorporated in the section “Climatic variables”:“These climatic variables were selected because some studies suggested that they have influence on guanaco demographic parameters: Rey et al. 201226 observed that after a severe drought the proportion guanaco yearlings/female decreased significantly, and Sarno et al. 199917 detected a negative effect of winter snowfall on guanaco juvenile survival; because we had insufficient snowfall data we used the winter temperature as a proxy for winter snowfall.” \"As authors stated in introduction, guanaco populations have undergone overhunting (poaching) and competition with sheep, these being two of the major forces of their dramatic decline in the last century. For that reason, it is quite surprising that no variables related to livestock were included in the modelling procedure. The population has increased a lot in the studied period (x8) and just climate and/or density-dependent process do not explain alone such increase in my view. Therefore authors should consider including livestock effects, its influence on resource availability along the studied period and the mentioned overhunting, as this could really explain the detected trend.\"In relation with the “poaching” the following paragraph was incorporated in section “Study area” to justify why this variable was not considered in our study:“In contrast to the continent, the puma (Puma concolor), the main predator of guanacos, is absent in the island; since 1977 the guanaco hunting has been controlled by Chilean National Forest Corporation (CONAF, according to its Spanish acronym) and the Chilean Agricultural and Livestock Service (SAG, according to its Spanish acronym).”In relation to the suggestion to incorporate livestock in the analysis, we re-analyzed our multiple regression incorporating the variable livestock as sheep population. We re-processed the regressions incorporating the sheep population as a new independent variable as a possible predictor of the guanaco population growth rate (l); so our new analysis increased from 5 to 6 independent variables: sheep population, guanaco population size, winter temperature, winter temperature with a lag of 1 year, annual precipitation, annual precipitation with a lag of 3 years. Our main results didn’t change and it was confirmed that only the guanaco population size seems to have an effect on the population growth rate (l). These changes can be found in the “Methods” and in the “Results” sections. “The guanaco sampling method has changed during the studied period (different band widths). Besides, were the transects chosen based on the topography and vegetation cover of the ranch to ensure that all areas were visible to census-takers along their transects? I am not sure if that could be a source of error, but authors should discuss this point.” In the Section “Guanaco population sampling” we added the following paragraphs where we explain the motive of the change and justify the transects chosen: “The sampling methodology was changed by an expert recommendation made in 1995 23; as in year 2000 the fixed and the undefined band width methods were applied simultaneously, we tested both results and found no significant statistical difference between them.”“Despite randomly selected transects are recommended24, preexisting roads were used because according to Soto (2010)18 the existing system of roads is an adequate sample of the area.” “Moreover, I recommend authors to re-analyze their data using the DISTANCE program (Buckland et al., 2004) to estimate population size over the period, as the analyses performed by this package seems much more robust that the one employed by authors. If they do so, authors could also include topographic variables in the models to see whether the resulting models change. The reason is that the authors do not explain if guanacos occur in both biomes (steppe and forest), because if that was true, detection of guanacos was completely different in both ecosystems. Therefore the band width of 500 or 1000m is not valid for forests.” In the Section “Guanaco population sampling” the following paragraphs was incorporated to justify the methodology used:“Raedeke (1978) 19 claims that the Leopold method is valid when the following conditions are satisfied: (1) the road systems must be an adequate sampling of the study area, (2) the network road must be randomly distributed, and (3) the animals included in the sampling must be randomly distributed in relation to the observer’s route; and he considers that in the south of the Tierra del Fuego Island these assumptions are fulfilled. Soto 201018 compared population estimations by the Leopold and Distance methods and found that the value of the means estimate by Leopold methods fall within the confidence intervals estimated by Distance; additionally, as all sampling periods used the same methodology whatever bias might exist in the estimation of the mean abundance using the Leopold method, the relative changes among years (and thus the temporal trend) will be adequately represented; thus, we conclude that the Leopold’s method seems adequate for our purposes18.”In relation to the forest and the sampling methodology, as the area occupied by forests is low and it has some degree of intervention, the visibility of the guanaco is adequate, hence we considered that the forest is a minor component and that does not affect the efficiency of the sampling procedure. In the section “Study area” the next paragraph was incorporated:“The forested area is about 8.8% of the total study area, with different degrees of forest clearance that offers adequate visibility for guanaco sampling.” “Regarding the population size estimated along the 36 years (Supplementary table), important (huge) differences are found, for example between 2004 and 2005 or between 2006 and 2007, or between 2007 and 2008. My concern is if such “jumps” could be related to the census surveyed. Please discuss this point.”In the “Discussion” section we added the following discussion as required by the reviewer:“The results of the guanaco population sampling suggest a certain trend in the population size, with a sort of exponential growth in the first few years, becoming more variable as the population grows, with more marked fluctuations during the last 10-12 years; this may imply that the population is becoming stabilized, possibly approaching its carrying capacity. On the other hand, the sampling results of those last 10-12 years show abrupt “jumps” in some years (for example 2004-2005) that may be the consequence of the mobility of guanacos from the Cameron ranch to neighboring sites and vice versa; since the transect sampling does not identify individuals the effects of possible local displacements could not be considered.” “The authors used a stepwise regression when other approaches such as logistic regressions, Ricker or Beverton-Holt models could give more suitable results. Please explain or justify your decision.”Our goal was to carry out a preliminary analysis that would guide us in the study of the population regulation process in the guanaco. As there are studies in ungulate that have used simple multiple regression analysis and obtained good/satisfactory results, we considered that it was of interest to test this technique as a first step in such an analysis.In the manuscript the following paragraph was incorporated in the “Discussion” section to justify our decision: \"Contrary to what was expected based on the bibliography of ungulate’s population dynamics, weather variables do not seem to influence the density-dependent population growth rate process of the guanaco population of the Cameron ranch. Our aim was to carry out a preliminary analysis that would help identify some of the regulation processes in guanaco population; so we used a simple multiple regression analysis as a first step in that direction; nevertheless, our conclusion is that in order to test the effects of climatic variables on population regulation of large ungulates as the guanaco, the use of a population dynamic model would be recommended, for they seem to account better for the interaction between density-dependent processes and weather variables than a simple regression between the latter and guanaco population growth rate.” “The selection of the best model among the candidates should be done by Akaike’s information”This suggestion was considered and the respective Akaike’s values were calculated. Minor comments:“Refer to climatic variables or climatic effects, instead of climatic covariables.”The recommendation was applied. “Explain more in detail Corani & Gatto’s studies.”With the previous changes we realize that the citation of Corani and Gatto (2007) is not necessary any more. Mainly because the essence of that paper is the proposal of a methodology that is not strictly relevant to our work. The phrase with this citation was deleted. “Please remove information related to metabolic studies between guanacos and sheep (1st paragraph of study area section).” The suggestion was carried out. “Please add Nothofagus spp among the brackets after deciduous forest (study area section) if it is the case.”This information is already incorporated. “Give more information about livestock ranching, density, and size and the relation of livestock rangers with guanacos along the studied period (study area section).”This information was already incorporate in the section “Sheep population” “Are the guanacos found both in the steppe and the forest biomes?” Yes, the guanacos use both biomes, but they mostly use the open habitat (i.e. steppes and meadows). “Please re-write the paragraph related to guanaco sampling through transect method in the consecutive periods (it is not clear enough).”The paragraph was improved. “Please remove information related to binoculars and GPS brands (did the authors use the same gadgets across 36 years?).”The recommendation was carried out. “Explain the interest of using time lags (T) as covariables in the model.”The next paragraph was incorporated in section “Population analysis”:“The inclusion of time lags is convenient because it may detect possible cumulative climatic effects on ungulate population growth rates as it was observed in, e.g., deer and moose 29 and in vicuñas13”. “Please add the Statistica version employed.”The Statistica tools was replaced by language R (Version 0.97.449 – ©2009-2012 RStudio, Inc.) to obtain the value of Akaike and satisfy the item 3.2 (see above). “Please move explanations of the analyses perform from results to material & methods section.”The recommendation was carried out." } ] } ]	1	https://f1000research.com/articles/2-210
https://f1000research.com/articles/3-132/v1	20 Jun 14	{ "type": "Research Article", "title": "Novel somatic single nucleotide variants within the RNA binding protein hnRNP A1 in multiple sclerosis patients", "authors": [ "Sangmin Lee", "Michael Levin", "Sangmin Lee" ], "abstract": "Some somatic single nucleotide variants (SNVs) are thought to be pathogenic, leading to neurological disease. We hypothesized that heterogeneous nuclear ribonuclear protein A1 (hnRNP A1), an autoantigen associated with multiple sclerosis (MS) would contain SNVs. MS patients develop antibodies to hnRNP A1293-304, an epitope within the M9 domain (AA268-305) of hnRNP A1. M9 is hnRNP A1’s nucleocytoplasmic transport domain, which binds transportin-1 (TPNO-1) and allows for hnRNP A1’s transport into and out of the nucleus. Genomic DNA sequencing of M9 revealed nine novel SNVs that resulted in an amino acid substitution in MS patients that were not present in controls. SNVs occurred within the TPNO-1 binding domain (hnRNP A1268-289) and the MS IgG epitope (hnRNP A1293-304), within M9. In contrast to the nuclear localization of wild type (WT) hnRNP A1, mutant hnRNP A1 mis-localized to the cytoplasm, co-localized with stress granules and caused cellular apoptosis. Whilst WT hnRNP A1 bound TPNO-1, mutant hnRNP A1 showed reduced TPNO-1 binding. These data suggest SNVs in hnRNP A1 might contribute to pathogenesis of MS.", "keywords": [ "Multiple sclerosis (MS) is the most common autoimmune disease of the central nervous system (CNS) in humans", "whose pathogenesis remains unknown. A number of genetic and immune studies indicate dysregulated immune responses as contributors to the pathogenesis of MS1–7. Genetic analyses show an association of MS with major histocompatibility complex (MHC) Class II human leukocyte antigen (HLA)-DRB-1 and single nucleotide polymorphisms (SNPs) related to immune function1", "2", "8. Both Th1/Th17 CD4+ T-lymphocytes and immunoglobulins appear to have a causative role1", "2", "9. Immunoglobulin G (IgG) responses to myelin and non-myelin targets have differentiated some MS patients from healthy controls9–11. Non-myelin antigens that are targets for immunoglobulins isolated from MS patients include neurofilaments", "axonal neurofascin and RNA binding proteins", "including heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1)9", "12–16." ], "content": "Introduction\n\nMultiple sclerosis (MS) is the most common autoimmune disease of the central nervous system (CNS) in humans, whose pathogenesis remains unknown. A number of genetic and immune studies indicate dysregulated immune responses as contributors to the pathogenesis of MS1–7. Genetic analyses show an association of MS with major histocompatibility complex (MHC) Class II human leukocyte antigen (HLA)-DRB-1 and single nucleotide polymorphisms (SNPs) related to immune function1,2,8. Both Th1/Th17 CD4+ T-lymphocytes and immunoglobulins appear to have a causative role1,2,9. Immunoglobulin G (IgG) responses to myelin and non-myelin targets have differentiated some MS patients from healthy controls9–11. Non-myelin antigens that are targets for immunoglobulins isolated from MS patients include neurofilaments, axonal neurofascin and RNA binding proteins, including heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1)9,12–16.\n\nRecently, mutations in RNA binding proteins have been shown to cause neurological disease17–21. For example, a mutation (p.D263V) in the prion-like domain (PrLD) of hnRNP A1 has been shown to cause familial amyotrophic lateral sclerosis (ALS)22. In addition to inherited mutations, somatic variants have also been shown to cause neurological disease23. hnRNP A1 performs a number of critical cellular functions related to transcription, nucleocytoplasmic transport of mRNA and translation24,25. In addition to the PrLD, other important functional domains in hnRNP A1 include two RNA binding domains (RBDs) and M9, its nucleocytoplasmic shuttling domain22. M9 binds its nuclear receptor, transportin-1 (TPNO-1, also known as karyopherin β2) and the hnRNP A1:TPNO-1 complex is transported into and out of the nucleus3,9,16,26,27.\n\nOur lab has performed extensive studies on the role of autoimmunity to hnRNP A1 in MS and human T-lymphotropic virus type 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis (HAM/TSP), a viral-induced model and clinical mimic of MS3,28–30. Initially, we discovered that HAM/TSP patients develop antibodies to hnRNP A1 that cross-react with HTLV-1-tax, indicative of molecular mimicry29,31. Next, the epitope of the HAM/TSP IgG response (AA293-304) was localized to M9 (AA268-305)32. M9 is a bipartite phenylalanine-tyrosine nuclear localization sequence (PY-NLS) that requires binding to TPNO-1 for hnRNP A1 to shuttle between the nucleus and cytoplasm16,31 Because of the similarities between MS and HAM/TSP, we hypothesized that MS patients would also develop antibodies to hnRNP A1. In fact, antibodies isolated from MS patients, in contrast to healthy controls and Alzheimer’s patients, were also found to immunoreact with the identical hnRNP-A1-M9 epitope (AA293-304)16. Subsequent studies indicated that the IgG was biologically active and potentially pathogenic. For example, mono-specific antibodies to hnRNP A1 isolated from patients caused decreased neuronal firing using neuronal patch clamp in rat brain sections31,33. Further, neurons exposed to anti-hnRNP A1-M9293-304 specific antibodies resulted in neurodegeneration and neuronal death16,34. The anti-hnRNP A1-M9293-304 specific antibodies also caused changes in neuronal RNA expression that correlate with the clinical phenotype of MS and HAM/TSP patients (ie. spastic paraparesis), which was subsequently confirmed in neurons isolated from the brains of MS patients16. Additional studies showed that anti-hnRNP A1-M9293-304 specific antibodies entered neurons via clathrin-mediated endocytosis and caused apoptosis in a neuronal cell line34. Anti-hnRNP A1-M9293-304 specific antibodies also caused a redistribution of hnRNP A1 in neurons from nuclear to an equal distribution of nuclear and cytoplasmic localization, suggesting the antibodies interfered with M9, which is required for hnRNP A1s nuclear import34. Considering: (1) the role of hnRNP A1 in cellular function; (2) variant forms of hnRNP A1 cause neurodegenerative disease, and (3) hnRNP A1 is an autoimmune target in MS patients, we hypothesized that MS patients would contain novel genomic DNA single nucleotide variants (SNVs) in hnRNP A1-M9, which when expressed, would alter cellular function and contribute to cell death.\n\n\nMethods\n\nAll blood samples were collected according to the approved Institutional Review Board protocols (Veterans Affairs Medical Center - Memphis, Study #317164, University of Tennessee Health Science Center, Study #98-06618-FB) with patient consent. The diagnosis of MS was made using published criteria (Supplement 1, see Results)35.\n\nHuman PBMCs were isolated from fresh blood by Ficoll-Paque gradient centrifugation and washed with PBS. Genomic DNA was isolated from PBMCs using the QIAmp blood kit (Quiagen Inc., Chatsworth, CA, U.S.A.) according to manufacturer's protocol. All DNA samples were quantified using Nanodrop (Quawell) and restriction enzyme digestion methods.\n\nSpecific oligonucleotides were designed from the published genomic DNA sequence of the human hnRNP A1 gene. The upstream primer 5′- CAGATAAAGGC CCTCTTTCCC -3′ (3080–3100) and the downstream primer 5′- CTCAGCTACATTAGGGTTATTGGG -3′ (3667–3690) flank a 611 bp region of the human hnRNP A1 genomic DNA containing exon 8 and exon 9.\n\nOne microgram of genomic DNA was amplified in a reaction mixture containing the primers and KOD Hot Start DNA polymerase (Novagen). Use of this DNA polymerase has a mutation frequency of 0.10%36. Before adding enzyme, the reaction mixture was heated at 95°C for 2 minutes. Amplification was carried out for 35 cycles of denaturation at 95°C for 20 s, annealing at 57°C for 10 s, and extension at 72°C for 15 s, followed by terminal elongation at 70°C for 20 s. The resulting PCR product was cloned into the pCR2.1-TOPO blunt vector (Invitrogen), yielding pCR2.1-TOPO-Blunt-hnRNP A1-611 bp, and E. coli TOP10 was transformed with this plasmid. Purified plasmid was digested with EcoRI yielding either one band (no insert) or two bands, 3.9 kbp (plasmid) and 611 bp (insert). Digests were subjected to electrophoresis on 1.5% agarose gel and visualized with the Gel Logic 200 Imaging System (Kodak). Clones that contained the 611 bp insert were sequenced (Supplement 3).\n\nTue pCR2.1-TOPO-Blunt-hnRNP A1 611 bp clones were subjected directly to automated DNA sequencing (ABI 3130 X L) at the University of Tennessee Health Sciences Center Molecular Resource Center. Electropherograms were obtained and sequence quality was analyzed by Sequence Analysis Software (ABI). Sequence alignment was carried out by Nucleotide BLAST (National Center for Biotechnology Information Called genomic DNA sequences were compared to mutations (SNVs, SNPs) listed in four different public databases: (1) Exome variant server (ESV): http://evs.gs.washington.edu/EVS/, (2) Catalogue of somatic mutations in cancer (COSMIC): http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/, (3) 1000 genomes; a deep catalog of human genetic variation: http://www.1000genomes.org, (4) NCBI dbSNP: (http://www.ncbi.nlm.nih.gov/snp/).\n\ncDNA encoding the entire sequence of hnRNP A1 (WT) was cloned into the expression vector pTriEx™5 Ek/LIC vector (Novagen) and transfected into SK-N-SH cells, a neuroblastoma cell line (ATCC - American Type Culture Collection). The amplified open reading frame (ORF) of hnRNP A1 was subcloned into Bam HI and Hind III sites of modified pGEX-6p-1 vector to create recombinant E. coli expression vectors for gluthathione S-transferase (GST) full down assay.\n\nThe primers for mutagenesis by PCR were designed basically according to the manufacturer (QuikChange™ II XL Site-Directed Mutagenesis kit; Agilent Technologies, CA). Briefly, each pair of primers contained a primer-primer complementary (overlapping) sequence at the 3′- and 5′-terminus. The designed primers were used for mutagenesis of the target residues F273L, M276L and F281L in hnRNP A1. The primers for each of the variants were: (1) p.F273L - forward: CAG TCT TCA AAT CTT GGA CCC ATG AAG GGA GG, reverse: CCT CCC TTC AGG GGT CCA AAA TTT GAA GAC TG; (2) p.M276L - forward: CAG TCT TCA AAT TTT GGA CCC CTG AAG GGA G, reverse: CCT CCC TTC ATG GGT CCA AGA TTT GAA GAC TG; (3) p.F281L - forward: C ATG AAG GGA GGA AAT CTT GGA GGC AGA AGC TC, reverse: GA GCT TCT GCC TCC AAG ATT TCC TCC CTT CAT G. All variant sites were located in hnRNPA1-M9 and both forward and reverse primers shared the region in question. The melting temperature (Tm) was calculated using the formula provided by the manufacturer Agilent Technologies: Tm = 81.5+0.41(%GC)-675/N-% mismatch. Here, N is the primer length in bases. All the primers were synthesized by Genelink (Hawthorne, NY). Mutagenic reaction was performed in 50 µl of PCR mix containing 10 ng of pTriEx-5 Ek/LIC-hnRNP A1(WT) or pGEX-6p-1-hnRNP A1(WT) as template, 200 nM primer and 2.5 U Pfu DNA polymerase. The PCR temperature profile was: an initial denaturation at 95°C for 1min, followed by 18 cycles with each at 95°C for 50 sec, 60°C for 50 sec and 68°C for 1 kb/min, and a final extension at 68°C for 7 min. The PCR products of Site-Directed Mutagenesis were transformed into E. coli XL10-Gold competent cells and isolated using Qiagen miniprep kits (Qiagen, Germany).\n\nDNA complexes prepared using a DNA (μg) to Lipofectamine® 2000 (μl) ratio of 1:2.5 for SK-N-SH cell line. For hnRNP A1 relocalization experiments, the human hnRNP A1 (WT or variant) cDNA was transfected into SK-N-SH cells (70–80% confluence) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. After 5 hours incubation, the transfection mixture was removed from each well and replaced with DMEM containing 10% FBS. Fresh medium was conditioned for 24 h before relocalization analysis of hnRNP A1 by immunocytochemistry.\n\nSK-N-SH Cells (ATCC HTB-11) were grown on poly-l-lysine-coated cover slips and were transfected using Lipofectamine 2000. Cells were then rinsed with PBS, fixed with 4% paraformaldehyde, permeabilized with cold acetone, and blocked in PBS containing 5% BSA. Primary antibodies used were: rabbit anti-TDP-43 (1:1000, Millipore, catalog #ABN271), rabbit anti-active caspase-3 (1:50, Millipore, catalog #AB3623), rabbit anti-Neuron specific beta III tubulin (NTB3) (1:1000, Abcam, catalog #ab18207) and biotinylated mouse anti-strep-Tag II (1:1000, GenScript, catalog #A01737). Secondary antibodies were: Texas Red conjugated goat anti-rabbit IgG (1:300, Vector, catalog #TI-5000 and FITC conjugated strepavidin (1:300, Vector, catalog #SA-5001). Primary antibodies were diluted in blocking solution incubated with each coverslip for overnight at 4°C. Cells were then washed with PBS and incubated in secondary antibody for 1 hr. Cells were then washed with PBS and mounted in Prolong-Gold anti-fade reagent with DAPI (Invitrogen).\n\nSK-N-SH cells were cultured in Dulbecco’s Modified Eagle’s medium (BD Biosciences) supplemented with 10% fetal bovine serum, 100 U/mL penicillin G and 100 μg/mL streptomycin, at 37°C under 5% CO2. Cells were harvested and lysed with CytoBuster™ Protein Extraction Reagent (Millipore), containing inhibitor cocktail, homogenized for a few seconds with a handheld homogenizer and spun at 16,000 × g for 5 minutes. Supernatants were used for GST-pull down assays. Glutathione-Sepharose 4B beads coupled with GST-hnRNP A1 (WT or variant), which includes the Transportin 1-binding domain, were incubated for 1 h at 4°C with 600 μL of the cell lysates in CytoBuster™ Protein Extraction Reagent and protease inhibitors. After washing the beads three times with 600 μL of 10 mM PBS (10 mM Na2HPO4, 140 mM NaCl, 2.7 mM KCl, 1.8 mM KH2PO4, pH 7.4) and protease inhibitors, proteins bound to the beads were analyzed by 8–16% SDS-PAGE followed by immunoblotting with rabbit polyclonal GST antibody (1:1000, Millipore, catalog #06-332), mouse monoclonal Transportin 1 antibody (1:1000, Millipore, catalog #05-1515) and mouse monoclonal TDP-43 antibody(1:1000, Millipore, catalog #MABN45). The immunoreactive bands were visualized using enhanced chemiluminescence.\n\n\nResults\n\nWe sequenced a 611 bp region of hnRNP A1 genomic DNA inclusive of exons 8 and 9 with intervening introns (NP_002127.1, Chromosome 12q13.1, DNA g.3080-3690, RNA (cDNA) c.752-963, protein AA252-320) (Figure 1A) isolated from the PBMCs of patients with MS: relapsing remitting MS (RRMS, n=5), secondary progressive (SPMS, n=5) and primary progressive MS (PPMS, n=4) and healthy controls (HC, n=6) (Supplement 1). The expressed sequence included: the C-terminal of the PrLD (AA252-267), M9 (AA268-305) and the residual C-terminus of hnRNP A1 (AA306-320) (Figure 1A). This region also includes the ‘core’ TPNO-1 binding domain (AA268-289) and the MS IgG epitope (AA293-304). Some literature indicates that the PrLD and TPNO-1 binding domain may overlap, such that the PrLD region includes AA233-272 and the TPNO-1 binding domain includes AA263-289, with the a resulting overlap of AA263-272 (Supplement 2)22,26. In addition, the previously reported mutations (p.D262V (familial), p.N267S (sporadic)) that cause ALS are also contained within the target sequence (Figure 1A, Supplement 2)22. A small percentage of clones from each individual contained genomic DNA SNVs, indicative of these being somatic SNVs derived from a small percentage or subset of PBMC. SNVs were compared to those found in four different databases (see Methods and Supplement 2).\n\nA: Domain schematic of hnRNP A1. The sequence shown is isoform A (NP_002127). Isoform B contains a 52 amino acid insert following AA251, resulting in a 372 amino acid protein (not shown). The RNA binding domains (RBD 1 and 2) are contained within the N-terminal half of hnRNP A1. The prion-like domain (PrLD, AA233-267), M9 (AA268-305) and the C-terminus (AA306-320) are shown. M9 (orange) contains the ‘core’ transportin-1 binding domain (TPNO-1, AA268-289, red) and the MS IgG epitope (AA293-304, yellow). Some literature indicates that the PrLD and TPNO-1 binding domain may overlap, such that the PrLD region includes AA233-272 and the TPNO-1 binding domain includes AA263-289, with the a resulting overlap of AA263-272 (Supplement 2). The amplicon included DNA from exons 8 and 9 with the intervening intron. The expressed protein included the PrLD that contained ALS-associated mutations (p.D262V (familial), p.N267S (sporadic)(blue)), as well as M9 and the C-terminus of hnRNP A1. (bp base pair). B: Sequence alignment of hnRNP A1-M9. Human sequences are 100% conserved in mammals, except for M. mulatta (rhesus). There is also high sequence conservation between orthologs (mammals, non-mammalian vertebrates (G. gallus - chicken, D. rerio - zebrafish, X. laevis - frog) and invertebrates (D. melanogaster - fruit fly). SNVs resulting in amino acid substitutions in the TPNO-1 core domain are highlighted in red boxes. SNVs resulting in amino acid substitutions in the MS IgG epitope are highlighted in yellow boxes. Colored amino acids are present for clarity identifying conserved sequences through all species. Black lines (gaps) are inserted between residues so that similar or identical amino acids are aligned in each column. (http://www.bioinformatics.org/strap/)\n\nOf the six HCs, zero SNVs resulted in an amino acid substitution within the TPNO-1 binding domain, MS IgG epitope or M9 from the 481 clones that were sequenced (Table 1, Supplement 2, Supplement 3). One individual had a likely benign variant which did not result in a change in the associated amino acid (c.900A>G, p.R300R) (Supplement 2), and three others had SNVs in the C-terminal region (c.922T>C, p.S308P; c.949G>A, p.G317S; c.952A>G, p.R318G), which altered the amino acid sequence (Table 1, Supplement 2, Supplement 3). The SNV at AA308 was previously reported and not associated with disease (http://www.ncbi.nlm.nih.gov/snp). These data, in which SNVs in hnRNP A1 are a rare event, are consistent with the finding in sporadic (1 of 305) and familial ALS (1 of 212) patients where most did not have mutations by whole exome sequencing22.\n\nhnRNP A1 single nucleotide variants that resulted in an amino acid substitution. HC: Healthy Control. RRMS: Relapsing Remitting Multiple Sclerosis. SPMS: Secondary Progressive Multiple Sclerosis. PPMS: Primary Progressive Multiple Sclerosis. TPNO-1: Transportin 1. PRLD: Prion-like Domain. AA: aminoacid. SNV: single nucleotide variant.\n\nOf the five RRMS patients in which 358 clones were sequenced, one patient had two novel SNVs contained within the TPNO-1 binding domain that resulted in an amino acid substitution (c.826A>C, p.M276L; c.839A>G, p.N280S) (Table 1, Supplement 2, Supplement 3, Data availability). None of the other RRMS patients had changes within the TPNO-1 binding domain or M9. Other SNVs that resulted in an amino acid substitution included those within the C-terminal region (c.937A>G, p.S313G; c.940T>C, p.Y314H; c.955A>G, p.R319G) and one within the PrLD (c.775A>G, p.S259G). These SNVs are also novel (Table 1, Supplement 2, Data availability). There was a single SNV (c.963A>G) that did not alter the stop codon amino acid sequence (Supplement 2).\n\nOf the five SPMS patients, one had a novel SNV that resulted in an amino acid substitution in the ‘core’ TPNO-1 binding domain (c.817T>G, p.F273L). Although there was a somatic SNV contained within this codon in the COSMIC database (in patients with cancer), its SNV and amino acid change were different (c.818T>G, p.F273C). A second patient had a novel SNV contained within the PrLD (c.755G>A, p.S252N), which also aligned with a somatic SNV in the COSMIC database (c.755G>T, p.S252I), but yielded different amino acids. A third patient had an SNV within the PrLD (c.787T>C, p.F263L), which also aligned with a somatic SNV contained within this codon in the COSMIC database (c.789T>G, p.F263L). In contrast to HC, RRMS or PPMS, novel SNVs that resulted in an amino acid substitution in SPMS predominated within the MS IgG binding epitope of M9 (c.884A>G, p.Y295C; c.886T>C, p.F296L; c896C>T, p.P299L; c.898C>T, p.R300S; c.902A>G, p.N301S) (Table 1, Supplement 2, Supplement 3, Data availability). Overall, of the 355 clones that were sequenced from the five SPMS patients, 8 (2.25%) were contained within the PrLD and M9 domains of which 6 were within M9, and 5 were exclusive to the MS IgG epitope (Table 1, Supplement 2, Supplement 3). Like HC and RRMS, there were several SNVs contained in the C-terminal region of hnRNP A1 (c.941A>G, p.Y314C; c.958T>C, p.F320L) (Table 1, Supplement 2, Supplement 3).\n\nIn contrast to HC, all four PPMS patients, had novel somatic SNVs that resulted in an amino acid substitution and were contained within the ‘core’ TPNO-1 binding domain of hnRNP A1 as follows: patient 1 (c.823C>T, p.P275S), patient 2 (c.831G>T, p.K277N), patient 3 (c.850A>G, p.R284G), and patient 4 (c.817T>C, p.F273L; c.841T>C, p.F281L; c.853A>G, p.S285G) (Table 1, Supplement 2, Data availability). Patients 1 and 3 each had a novel SNV within the PrLD region (c.793A>G, p.N265D). Thus, of the 317 clones that were sequenced, 2.84% were contained within the M9 or PrLD domains, two-thirds of which were exclusive to the TPNO-1 binding domain (Supplement 3). Other SNVs were contained within the MS IgG binding epitope (c.901A>G, p.N301D) or the C-terminal region (c.922T>G, p.S308P; c.950G>A, p.G317D) (Table 1, Supplement 2, Supplement 3, Data availability). Only the c.817T>C, p.F273L SNV aligned with a somatic SNV within the same codon in the COSMIC database (c.818T>G, p.F273C), but again, both the SNV and amino acid substitution differed.\n\nThe TPNO-1 binding domain is highly conserved within mammals and evolutionarily conserved between species (Figure 1B). Specifically, the TPNO-1 binding domain is 100% conserved in mammals, except for five mutations contained only within the rhesus monkey (Macaca mulatta) (only one of which overlapped with the SNVs we discovered (AA300) (Figure 1B)). Further, amino acid sequences were highly conserved between species, as shown by the orthologs between mammals, bird (Gallus gallus), fish (Danio rerio) and frog (Xenopus laevis) as well as with the fruit fly (Drosophila melanogaster) (Figure 1B). Taken together, these data indicate that variants in this highly conserved domain may have pathological consequences, which might contribute to human disease.\n\nA total of nine novel SNVs that resulted in an amino acid substitution were discovered in MS patients within the ‘core’ TPNO-1 binding domain of hnRNP A1-M9. hnRNP A1 has a number of functions, including the transport of nascent mRNA from the nucleus to the cytoplasm. hnRNP A1 shuttles between the nucleus and cytoplasm and binds TPNO-1, which is required for its nuclear import16,26,27. At equilibrium, hnRNP A1 is predominantly found in the nucleus37,38. Considering the disease-associated SNVs are contained within a highly conserved region of hnRNP A1 that plays a critical role in cellular function, we hypothesized that altered hnRNP A1 would change hnRNP A1 localization, TPNO-1 binding and induce cellular damage. To test this hypothesis, we performed a number of experiments. First, we manufactured three different hnRNP A1 mutations (by site-directed mutagenesis) contained within its TPNO-1 binding domain (F273L, M276L and F281L), transfected each mutant into SK-N-SH cells and examined the cells for hnRNP A1 localization relative to transfection of Wild Type (WT) hnRNP A1. As shown in Figure 2A (upper panel), WT hnRNP A1 almost completely localized to the nucleus of SK-N-SH cells. In contrast, mutant forms of hnRNP A1 localized to the cytoplasm of cells (Figure 2A, lower panel). Localization within the cytoplasm was not diffuse, but granular, suggestive of stress granule (SG) formation (Figure 2A, lower panel, arrows). There was also localization within cellular processes (Figure 2A, lower panel, arrowhead). To confirm that mutant hnRNP A1 was present in SGs, we double-labeled SK-N-SH cells that contained the transfected mutant hnRNP A1 with anti-TDP-43 antibodies. As shown in Figure 2B, like hnRNP A1, TDP-43 was localized to nuclei (without transfection). In addition, WT hnRNP A1 and TDP-43 co-localized within the nuclei of SK-N-SH cells. In contrast, mutant hnRNP A1 (F273L, M276L and F281L) co-localized with TDP-43 within the cytoplasm of cells (Figure 2B). Considering recent data indicating binding between hnRNP A1 and TDP-43; co-localization of mutant hnRNP A1 (p.D262V) to TDP-43 containing SGs; the role of TDP-43 in SG formation, and the localization of hnRNP A1 in SGs in stress activated cells, these experiments confirm that mutant hnRNP A1 is contained within TDP-43 positive SGs22,39–42. Because TPNO-1 is required for hnRNP A1 nucleocytoplasmic transport, we hypothesized that mutant hnRNP A1 would alter its binding to TPNO-1. In these experiments, protein lysates purified from SK-N-SH cells were incubated with either WT or mutant GST-tagged-hnRNP A1 bound to Glutathione-Sepharose 4B beads. The resultant eluent was then probed for TPNO-1. As shown in Figure 3A, western blots showed immunoreactivity for TPNO-1 protein with WT-hnRNP A1, indicative of TPNO-1’s binding to hnRNP A1. In contrast, there was significantly reduced binding between mutant forms of hnRNP A1 and TPNO-1 (Figure 3A). These experiments show that mutations in the TPNO-1 binding domain of hnRNP A1-M9 alter TPNO-1 binding to hnRNP A1. To confirm protein binding between hnRNP A1 and TDP-43, which were visualized by immunocytochemistry (Figure 2B), we probed the identical eluents with an anti-TDP-43 antibody. Both WT and mutant hnRNP A1 bound TDP-43 (Figure 3B), indicative of their interaction in both the nuclei and cytoplasmic SGs in the cell line. This is consistent with other reports indicative of an interaction between hnRNP A1 and TDP-4322.\n\nA. Localization of WT and F281L hnRNP A1. (upper panel) hnRNP A1 localizes to the nuclei of cells transfected (T) with WT hnRNP A1. Untransfected (U) cells are also present in the same field. (Lower panel) In contrast, mutant hnRNP A1 (F281L) mis-localizes to the cytoplasm including cellular processes (arrowhead) in a granular pattern, consistent with stress granule (SGs) formation (arrows) (NTB3-Neuron specific beta III tubulin). B. Co-localization of hnRNP A1 and TDP-43. TDP-43 localizes to nuclei of neurons (‘no tx’) (pink or red signal). WT hnRNP A1 co-localizes with TDP-43 in nuclei (arrows). In contrast, mutant forms of hnRNP A1 (F273L, M276L, F281L) predominantly mis-localize to the cytoplasm and co-localize with TDP-43 positive SGs (arrows, yellow). Some TDP-43 remains in the nucleus (pink or red signal). Figures 2C and D: Apoptosis caused by mutant hnRNP A1 in SK-N-SH neurons. C. Transfection of mutant hnRNP A1 (F273L, F281) result in apoptotic blebs in transfected cells (T) (arrows), in contrast to untransfected (U) cells in the same field under identical conditions. D. Transfection of WT hnRNP A1 localized to the nucleus and showed no evidence of active caspase-3 labeling. In contrast, mutant hnRNP A1 (F273L, M276L, F281L) predominantly localized to the cytoplasm of cells that stained with active caspase-3, many of which with fragmented nuclei (arrowheads), both of which are indicative of apoptosis. There was also co-localization of active caspase-3 and mutant hnRNP A1 (arrow).\n\nGST-tagged WT or mutant forms of hnRNP A1 were bound to Glutathione Sepharose 4B beads, protein lysates of SK-N-SH cells were applied to columns, and the resulting eluent was probed for either TPNO-1 (A) or TDP-43 (B). A. There was strong binding between WT hnRNP A1 and TPNO-1. In contrast, there was no binding between mutant hnRNP A1 (F273L) and TPNO-1, and markedly reduced binding between mutant hnRNP A1s (M276L, F281L) and TPNO-1. B. There was no change in binding between WT and mutant forms of hnRNP A1 and TDP-43.\n\nIn the in-vitro experiments, SG formation in SK-N-SH cells formed within several hours of transfection. When we waited overnight (approximately 24 hours) the cells containing mutant hnRNP A1 developed apoptotic blebs, which contained hnRNP A1 (Figure 2C, arrows). Apoptosis was confirmed by active caspase-3 staining (Figure 2D). As shown in Figure 2D, SK-N-SH cells transfected with mutant hnRNP A1 showed a cytoplasmic hnRNP A1 distribution, stained positive for active caspase-3 and contained fragment nuclei, confirming apoptosis.\n\nIn summary, in contrast to WT hnRNP A1, mutant hnRNP A1 showed markedly reduced binding to its co-receptor TPNO-1, co-localized with TDP-43 within cytoplasmic SGs of cells and caused apoptosis, indicative of the potential pathogenic nature of these disease-associated SNVs in MS patients.\n\n\nDiscussion\n\nRecent studies indicate that in addition to cancer, somatic variants can cause neurological disease23. In this study, we discovered novel somatic genomic DNA SNVs that have the potential to contribute to the development of MS. Nine were contained within the ‘core’ TPNO-1 binding domain of hnRNP A1-M9 (AA268-289). Three additional SNVs (c.793A>G, p.N265D (in two patients); c.787T>C, p.F263L) included amino acids within the PrLD - M9 overlap region (AA263-267), which also bind TPNO-145. These variants were in a region of hnRNP A1 that are adjacent to mutations shown to cause ALS (p.D262V, p.N267A). Interestingly, 8 of these 12 SNV’s that involved hnRNP A1-M9 binding to TPNO-1 occurred in PPMS patients. In addition, two hnRNP A1 SNVs were contained exclusively within the PrLD (c.755G>A, p.S252N; c.775A>G, p.S259G). There were also six novel SNVs that resulted in an amino acid substitution within the MS IgG epitope of M9 (AA293-304), five of which segregated to patients with SPMS. Finally, there were nine SNV’s in the C-terminal of hnRNP A1 (AA306-320), occurring with similar frequency in HCs and MS patients. The overall somatic SNV rate (based on the number of clones sequenced) for the M9 target sequence was: PPMS - 2.21%, SPMS - 1.69%, RRMS - 0.56%, HC - 0%. If one includes the PrLD (a domain shown to be critical to hnRNP A1 function), the rates increase in PPMS, SPMS and RRMS to 2.84%, 2.25% and 0.84% respectively. None were identical to somatic mutations in the COSMIC database (n = 981,720 samples, n = 1,292,597 unique variants). We utilized a PCR - cloning technique that has been fine-tuned for more than a decade and shows a mutation rate of approximately 0.1% in more than 46,000 clones that were examined36. The rates in progressive MS patients exceed this error rate by more than a log. In addition, under identical conditions, there were no mutations in the M9 target sequence or the PrLD domain in the HCs we examined. Thus, these results are unlikely to be due to PCR errors. Importantly, there was little or no overlap with either SNVs or SNPs reported in four different databases.\n\nhnRNP A1 was one of the first RNA binding proteins shown to shuttle into and out of the nucleus37,38. Nucleocytoplasmic transport is dependent upon binding between the M9 domain (AA268-305) of hnRNP A1 to TPNO-1, in order for this complex to pass through the nuclear pore. M9 acts as both an NES and NLS. M9 is a bipartite PY-NLS whose three-dimensional structure and binding contacts with TPNO-1 are well characterized26,27,43. Specifically, M9 contains three binding epitopes (Table 2): a hydrophobic (273-FGPM-276) domain, a basic residue (522R) and a C-terminal RX2-5 PY motif, each connected by ‘linker’ residues26,27,43. Each epitope, as well as individual amino acids within an epitope, conveys varying degrees of binding activity to TPNO-126,43,44. For example, mutant P275A dramatically inhibits nucleocytoplasmic transport and substitution of 273-FGPM-276 with 273-AAAA-276, completely abolishes TPNO-1 binding and nucleocytoplasmic transport37,43,44. Our data closely align with these findings. For instance, genomic SNVs in MS patients occurred at F273L, F275S, and M276L - all contained within epitope 1. Experimentally, we showed that transfection of F273L and M276L mutants caused mis-localization of hnRNP A1, SG formation (co-localizing with TDP-43), cellular apoptosis and diminution of TPNO-1 binding. Mutant F281L caused similar results. Interestingly, F273, F281 and R284 all have two or more side chains that bind TPNO-1, thus are critical contact points between M9 and TPNO-126. MS patients also had an SNV at R284G. Although we did not test this variant, a parallel substitution in the RNA binding protein fused in sarcoma (FUS) (R522G) (Table 2) caused a five-fold decrease in TPNO-1 binding and cytoplasmic mis-localization of FUS27. Like hnRNP A1, FUS is an hnRNP, which at equilibrium localizes to the nucleus and contains a bipartite PY-NLS that binds TPNO-127. Interestingly, mutations in FUS have been shown to cause ALS and frontotemporal lobe dementia17.\n\nBinding epitopes of the PY-NLS’s of hnRNP A1 and fused in sarcoma (FUS). Aligned PY-NLS sequences of hnRNP A1-M9 and FUS-NLS are shown. The three binding epitopes of the PY-NLS are shaded yellow (epitope 1 - hydrophobic), blue (epitope 2 - basic) and red (epitope 3 - C-terminal)27,43. SNVs resulting in an amino acid substitution (hnRNP A1) and missense mutations (FUS) are shown by gray shading of the amino acid sequence number. Amino acid SNVs associated with multiple sclerosis (MS) in hnRNP A1 are shown above the primary sequence and mutations associated with amyotrophic lateral sclerosis (ALS) in FUS are shown below the primary sequence. Two SNVs () in hnRNP A1-M9 align with mutations in the FUS NLS: hnRNP A1-M9 AA275 with FUS NLS AA510 and hnRNP A1-M9 AA284 with FUS NLS AA522. Stop codons, frame shift mutations, insertions and deletions not included. Notes: 1R514G/S, 2R514S, G515C, 3R514S, E516V17.\n\nImportantly, none of the SNVs contained within hnRNP A1 - M9 or the PrLD has been reported previously. For decades, the only certain genetic risk factor for MS was with MHC Class II HLA-DRB18. Genome Wide Association Studies (GWAS) have uncovered novel genetic associations with MS1,2,45 including with the interleukin-2 receptor-α and interleukin-7 receptor genes45. Subsequent studies using several thousand MS cases and controls, which analyzed hundreds of thousands of autosomal SNPs, confirmed the association of MS with major MHC Class II HLA-DRB1 (DRB1 15:01, 15:03, 13:03) and the protective effect MHC Class I HLA-A*021,8. Additional studies showed a total of 48 new and 49 known non-MHC SNPs associated with MS2. Interestingly, the functions of the vast majority of the SNP’s were related to CD4+ T-lymphocyte and immune regulation1,8. This is important, considering the role that T-lymphocytes and the immune response play in the pathogenesis of MS. A few were potentially associated with neurodegeneration46. Further, >95% of the SNPs were intronic or intergenic, with only a few SNPs involving exons, in contrast to the somatic SNVs discovered here1,2. In addition to GWAS, whole exome sequencing (WES) is being used to examine differential gene expression in MS patients. In contrast to GWAS, which detects known SNPs and utilizes statistical analyses designed to reveal common variants, WES is designed to discover novel, rare pathologic variants8. One of the genes identified by GWAS was CYP27B1, which encodes an enzyme of the same name that converts 25-hydroxyvitamin D to 1,25 hydroxyvitamin D, the biologically active form of vitamin D1,8. A single individual from 43 MS families was found to have a rare p.R389H genetic variant in CYP27B1, which resulted in complete loss of enzyme activity8,47,48. However, unaffected relatives of the individual also carried the identical variant, which has a high frequency in the general population48.\n\nThus, it is clear that an individual’s genetic background makes an important contribution to the pathogenesis of MS. This supports the tripartite hypothesis that an environmental trigger in a genetically susceptible individual causes an autoimmune response to CNS antigens that result in the pathology observed in the brain and spinal cord of MS patients. Potential environmental triggers include viral infection, low vitamin D levels and sun exposure49–51. During the relapsing phase of MS, Th1 and Th17 CD4+ T-lymphocyte responses appear to predominate and correlate with focal MS plaque formation in the CNS5,52,53. As MS evolves into a secondary progressive phase, CNS damage becomes more diffuse5,6,53. Immune cells also become more diffuse and IgG containing plasma cells, B-lymphocytes and macrophage/microglia response predominate7,53. Many of these latter features are also characteristic of primary progressive MS7. How might variants in hnRNP A1 contribute to the pathogenesis of MS? Although direct evidence is not yet available and requires further study, the molecular consequences of abnormal forms of hnRNP A1 on cellular function may have profound effects on the immune system. For example, as it relates to the environment, hnRNP A1 regulates the synthesis of several viruses including human immunodeficiency virus, HTLV-1, and human rhinovirus54–56. Immunologically, hnRNP A1’s nucleocytoplasmic shuttling and RNA binding specificity is required for myelopoiesis and modulation of immune-mediated programmed cell death57–59. Further, apoptotic blebs (which we showed in this study to contain hnRNP A1) have profound effects on the immune response, as they are believed to initiate and perpetuate autoimmune diseases such as systemic lupus erythematosus60,61.\n\nIn addition, genetic mutations in hnRNP A1 alter neuronal function. For example, an inherited mutation in the PrLD of hnRNP A1 resulted in its mis-localization to TDP-43 positive cytoplasmic SGs and thus is thought to cause familial ALS22. A second mutation in the PrLD was also discovered in a sporadic case of ALS22. Interestingly, in addition to inherited and germ line mutations (including SNPs), recent data indicate that acquired mutations cause neurological disease23. For example, both familial (inherited) and acquired (‘de novo’, somatic variants) of doublecortin cause subcortical band heterotopia (SBH), a neuronal migration syndrome that results in epilepsy and intellectual disability62. In contrast to inherited mutations, somatic mutations may only be present in specific cell lineages23. In SBH, somatic mutations are only found in the DNA of neurons and lymphocytes, and not in other tissues (a ‘mosaic’)23,62. To date, we have only discovered somatic mutations in PBMCs of MS patients. Future studies will address their presence in CNS tissues of MS patients. However, if present in the CNS, abnormal forms of hnRNP A1 have profound effects on neurons, and thus might contribute to neurodegeneration present in MS, particularly in PPMS where the majority of the SNVs were located.\n\nIn summary, we discovered novel SNVs in MS patients. The SNVs involve the M9 nucleocytoplasmic binding domain of hnRNP A1, which when transfected into a cell line, resulted mis-localization of hnRNP A1 to cytoplasmic stress granules and cellular apoptosis. Future studies are required to replicate this data, expand it to include greater numbers of MS patients and perform functional studies of the SNVs in immune and nervous system cells of MS patients.\n\n\nData availability\n\nSingle nucleotide variants (SNVs) (somatic) in MS patients submitted to dbSNP63", "appendix": "Author contributions\n\n\n\nSL and MCL conceived the study. SL and MCL designed the experiments. SL carried out the bench research. MCL prepared the first draft of the manuscript. SL and MCL contributed to the experimental design and preparation of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nDrs. Michael Levin and Sangmin Lee have a United States patent pending titled “Biomarker for neurodegeneration in neurological disease”.\n\n\nGrant information\n\nThe project was supported by Award Number I01BX001996 from the Biomedical Laboratory Research and Development Service of the VA Office of Research and Development to Dr. Levin.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThis work is based upon work supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs and the Multiple Sclerosis Research Fund at the University of Tennessee Health Science Center.\n\n\nSupplementary materials\n\nSupplement 1: Patient demographics (xlsx)\n\nSupplement 2: Single nucleotide variants in the target sequence of hnRNP A1 (xlsx)\n\nSupplement 3: Number of clones containing single nucleotide variants (xlsx)\n\n\nReferences\n\nSawcer S, Hellenthal G, Pirinen M, et al.: Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBogerd HP, Benson RE, Truant R, et al.: Definition of a consensus transportin-specific nucleocytoplasmic transport signal. J Biol Chem. 1999; 274(14): 9771–7. PubMed Abstract \| Publisher Full Text\n\nHafler DA, Compston A, Sawcer S, et al.: Risk alleles for multiple sclerosis identified by a genomewide study. N Engl J Med. 2007; 357(9): 851–62. PubMed Abstract \| Publisher Full Text\n\nBush WS, McCauley JL, DeJager PL, et al.: A knowledge-driven interaction analysis reveals potential neurodegenerative mechanism of multiple sclerosis susceptibility. Genes Immun. 2011; 12(5): 335–40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRamagopalan SV, Dyment DA, Cader MZ, et al.: Rare variants in the CYP27B1 gene are associated with multiple sclerosis. Ann Neurol. 2011; 70(6): 881–6. PubMed Abstract \| Publisher Full Text\n\nOksenberg JR, Hauser SL: Decoding multiple sclerosis. Ann Neurol. 2011; 70(6): A5–7. PubMed Abstract \| Publisher Full Text\n\nMunger KL, Levin LI, Hollis BW, et al.: Serum 25-hydroxyvitamin D levels and risk of multiple sclerosis. JAMA. 2006; 296(23): 2832–8. PubMed Abstract \| Publisher Full Text\n\nAscherio A, Munger KL: Environmental risk factors for multiple sclerosis. Part I: the role of infection. Ann Neurol. 2007; 61(4): 288–99. PubMed Abstract \| Publisher Full Text\n\nAscherio A, Munger KL, Simon KC: Vitamin D and multiple sclerosis. Lancet Neurol. 2010; 9(6): 599–612. PubMed Abstract \| Publisher Full Text\n\nKleinewietfeld M, Manzel A, Titze J, et al.: Sodium chloride drives autoimmune disease by the induction of pathogenic TH17 cells. Nature. 2013; 496(7446): 518–22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLassmann H, van Horssen J, Mahad D: Progressive multiple sclerosis: pathology and pathogenesis. Nat Rev Neurol. 2012; 8(11): 647–56. PubMed Abstract \| Publisher Full Text\n\nNajera I, Krieg M, Karn J: Synergistic stimulation of HIV-1 rev-dependent export of unspliced mRNA to the cytoplasm by hnRNP A1. J Mol Biol. 1999; 285(5): 1951–64. PubMed Abstract \| Publisher Full Text\n\nKress E, Baydoun HH, Bex F, et al.: Critical role of hnRNP A1 in HTLV-1 replication in human transformed T lymphocytes. Retrovirology. 2005; 2: 8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCammas A, Pileur F, Bonnal S, et al.: Cytoplasmic relocalization of heterogeneous nuclear ribonucleoprotein A1 controls translation initiation of specific mRNAs. Mol Biol Cell. 2007; 18(12): 5048–59. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIervolino A, Santilli G, Trotta R, et al.: hnRNP A1 nucleocytoplasmic shuttling activity is required for normal myelopoiesis and BCR/ABL leukemogenesis. Mol Cell Biol. 2002; 22(7): 2255–66. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHamilton BJ, Burns CM, Nichols RC, et al.: Modulation of AUUUA response element binding by heterogeneous nuclear ribonucleoprotein A1 in human T lymphocytes. The roles of cytoplasmic location, transcription, and phosphorylation. J Biol Chem. 1997; 272(45): 28732–41. PubMed Abstract \| Publisher Full Text\n\nRajani DK, Walch M, Martinvalet D, et al.: Alterations in RNA processing during immune-mediated programmed cell death. Proc Natl Acad Sci U S A. 2012; 109(22): 8688–93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRadic MZ, Shah K, Zhang W, et al.: Heterogeneous nuclear ribonucleoprotein P2 is an autoantibody target in mice deficient for Mer Axl, and Tyro3 receptor tyrosine kinases. J Immunol. 2006; 176(1): 68–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWickman G, Julian L, Olson MF: How apoptotic cells aid in the removal of their own cold dead bodies. Cell Death Differ. 2012; 19(5): 735–42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBahi-Buisson N, Souville I, Fourniol FJ, et al.: New insights into genotype-phenotype correlations for the doublecortin-related lissencephaly spectrum. Brain. 2013; 136(Pt 1): 223–44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDatabase of Single Nucleotide Polymorphisms (dbSNP). Bethesda (MD): National Center for Biotechnology Information, National Library of Medicine. dbSNP accession:[ss1056389899 - ss1056389924], (dbSNP Build ID: [141]). Reference Source" }	[ { "id": "5210", "date": "03 Jul 2014", "name": "Hans Lassmann", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a potentially interesting study, reporting increased presence of novel somatic single nucleotide variants (SNVs) within the RNA binding protein hnRNP A1 in peripheral blood mononuclear cells (PBMCs) in patients with multiple sclerosis (MS) in comparison to controls. The disease associated SNVs resulted in a mis-localization of hnRNP A1 into cytoplasmic stress granules and this mis-localization was associated with cellular apoptosis in cells, transfected with the respective mutants. From a technical point of view the study is well performed. The question, however, remains what the findings mean for the pathogenesis of multiple sclerosis.It is interesting to see that there is apparently a high incidence of induced SNVs in PBMCs of MS patients, which most likely not only affects the gene investigated in this study. This may be due to the fact that MS is a chronic inflammatory disease, leading to proliferation of leukocytes (in particular T-lymphocytes) in the peripheral immune system. In addition increased oxidative stress in PBMCs of MS patients has been reported before. Both conditions may lead to increased somatic mutation in these cell populations. Thus, in a study like this the inclusion of a control population with long-lasting chronic inflammation would be important in addition to the study of normal controls. According to the presented data, the particular SNVs are also likely to result in cell elimination. Whether this changes the immune response in MS patients has not been investigated in this study.Finally the authors discuss that their findings may have implications for the understanding the mechanisms of tissue injury and neurodegeneration in MS. To support this conclusion, evidence has to be provided that similar somatic mutations are also present in cells of the central nervous system, such as neurons or oligodendrocytes. The basic observation of an increased incidence of somatic mutations in PBMCs in MS presented in this article is a first (but preliminary) observation, which is interesting.However, it is not clear what this means for the disease. The key message from the authors that this is an important finding for MS pathogenesis is not substantiated by the data. I agree with the authors that further experiments (possibly beyond the scope of a new version of this article) are needed. For example:To look for somatic mutations for other genes, to see whether the paper just shows that in PBMCs of MS patients there is an increased rate of somatic mutations in many different genes or whether the mutations in the specific gene are MS specific. To expand the sample of control patients, in particular by including patients with other chronic inflammatory diseases (e.g. patients with chronic rheumatoid arthritis). To search for similar mutations in neurons and glia of the MS brain to establish a link to neurodegeneration in this disease.", "responses": [ { "c_id": "975", "date": "11 Sep 2014", "name": "Michael Levin", "role": "Author Response", "response": "We appreciate Professor Lassmann’s comments including that the study was “well performed” and the acknowledgement that “increased incidence of somatic mutations in PBMCs in MS presented in this article is a first…”. We agree with his constructive critiques, which we have addressed below.To look for somatic mutations for other genes, to see whether the paper just shows that in PBMCs of MS patients there is an increased rate of somatic mutations in many different genes or whether the mutations in the specific gene are MS specific.Our ongoing studies are designed to evaluate more genes. Using next-generation sequencing, which is beyond the scope of this paper, we may be able to assess a broader number of genes, potentially including coding exomes of the human genome. In this study, we evaluated three different regions of hnRNP A1: the C-terminal of PrLD, M9 and hnRNP A1’s C-terminus. Within this subset of genes, we saw a segregation of SNVs. For example, there were similar numbers of SNVs in hnRNP A1’s C-terminus in all groups examined (Table 1, Supplement 2). In contrast, in PPMS SNVs segregated to the TPNO-1 binding domain of M9 and SPMS to the MS IgG epitope of M9. Although more genes are required to assess their role in MS compared to other diseases, in this study, we have already observed a differential expression of SNVs between types of MS and HCs. This is addressed in the last paragraph of the discussion. To expand the sample of control patients, in particular by including patients with other chronic inflammatory diseases (e.g. patients with chronic rheumatoid arthritis).We will add patients with other chronic inflammatory diseases such as rheumatoid arthritis to our future studies. Fortunately, there is a large rheumatoid arthritis research group at our institution, which allow access to these samples. We addressed this in the last paragraph of the discussion. To search for similar mutations in neurons and glia of the MS brain to establish a link to neurodegeneration in this disease.Our ongoing studies will apply the techniques used in this manuscript in central nervous system tissues isolated from MS patients at autopsy. This is addressed in paragraph 6 of the discussion. As we complete these studies, as well as studies examining the role of SNVs on the immune response in MS patients, this will address Professor Lassmann’s concern about these findings being relevant to the pathogenesis of MS. Professor Lassmann also suggested that oxidative stress might be a cause of SNVs in the PBMCs of MS patients. We added this insight with references to the fourth paragraph of the discussion: “MS is also characterized by increased oxidative stress (in PBMC and brain), which can cause DNA damage and somatic mutations.”" } ] }, { "id": "5207", "date": "23 Jul 2014", "name": "Michael K. Racke", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript by Lee and Levin continues work by Levin on the role of an immune response to hnRNP-A1 in human demyelinating disorders, including multiple sclerosis. Previously, Levin had shown that an antibody response to HTLV-I Tax cross-reacted with hnRNP-A1. Now, they go on to show that there are mutations in hnRNP-A1 that affect the localization of this protein and how it could affect neuronal survival in multiple sclerosis.In general, the experiments appear to be well performed. One might be interested to know whether testing in another cell line such as a oligodendroglial cell line would have similar effects on cell survival as in the neuroblastoma cell line. This could also be highly relevant in MS, as in addition to neuronal loss, there is demyelination and loss of oligodendrocytes.This work attempts to address whether mutations in hnRNP-A1 could contribute to MS pathogenesis. It would be interesting to note whether antibody responses to the protein correlated with mutations (i.e. does the mutation in the protein affect tolerance). Another issue that needs to be addressed is whether the mutations in the hnRNP-A1 gene are due to increased mutational frequency that can be observed in replicating cells, similar to the observation of increased hprt mutations made by Allegretta many years ago in MBP-specific T cells.Overall, this is an interesting study which certainly increases the interest in non-myelin targets in diseases such as MS.", "responses": [ { "c_id": "976", "date": "11 Sep 2014", "name": "Michael Levin", "role": "Author Response", "response": "We appreciate Professor Racke’s critique, including that the studies were “well performed.” The following concerns expressed by Professor Racke were addressed:One might be interested to know whether testing in another cell line such as a oligodendroglial cell line would have similar effects on cell survival as in the neuroblastoma cell line. This could also be highly relevant in MS, as in addition to neuronal loss, there is demyelination and loss of oligodendrocytes.We used SK-N-SH neurons as a model system to examine the effect that mutant forms of hnRNP A1 might have on target cell function. Considering that cells other than neurons are clearly involved in the pathogenesis of MS, we plan to add oligodendrocyte cell lines to future studies of mutant hnRNP A1. We addressed this in the sixth paragraph of the discussion. This work attempts to address whether mutations in hnRNP-A1 could contribute to MS pathogenesis. It would be interesting to note whether antibody responses to the protein correlated with mutations (i.e. does the mutation in the protein affect tolerance).We agree that this would be in important and insightful experiment, one that we think is beyond the scope of the current study. Our previous work showed that MS patients develop antibodies to hnRNP A1 using Western blots. We have not yet developed the technology that would allow us to quantitate the amount of antibodies to hnRNP A1 in individual patients. We will consider this in the future as we examine the potential connection between antibodies to and somatic mutations within hnRNP A1. Another issue that needs to be addressed is whether the mutations in the hnRNP-A1 gene are due to increased mutational frequency that can be observed in replicating cells, similar to the observation of increased hprt mutations made by Allegretta many years ago in MBP-specific T cells.We appreciate this insight offered by Professor Racke, which is very relevant to our work and have included it in paragraph 4 of the discussion." } ] }, { "id": "5807", "date": "10 Sep 2014", "name": "Elliot M. Frohman", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMy distinguished colleagues from the University of Tennessee in Memphis have performed an innovative set of complex studies seeking to elucidate somatic mutation variants in patients with MS; across the various clinical subtypes, and compared to normal subjects. Quite surprising to this reviewer, the authors identified a TPNO-1 binding domain change of hnRNPA1-M9; an RNA binding protein that is among the first to be shuttled into and out of the nucleus. Perhaps even more conspicuous is that these somatic modifications are directly adjacent to well characterized mutations that are associated with the development of ALS (i.e. accelerated motor neuron disease of the upper and lower motor neuron compartments). My colleagues further show that 8/12 SNVs are shown to occur in primary progressive MS (a slowly and insidiously progressive loss of the upper motor neuron compartment of the CNS. They further elucidated six novel SNVs; with substitutions within the MS IgG epitope of M9 (AA 293-304).The authors proceed to provide a SNV rate stratification across normal subjects vs clinical subtypes of MS. Here they found the following corresponding rates:PPMS- 2.21%SPMS- 1.69%RRMS- 0.56%Normal human subjects = 0%Much has been achieved with the application of genome wide association studies (GWAS), such as the identification of a linkage between the IL-2 and IL-7 receptor genes; immune regulatory elements that dovetail nicely with the higher risk of MS developing in subjects who are also positive for the MHC Class II HLADR21501B1 (2 copies > 1 copy with respect to relative risk). Alternately, MHC Class I HLA-A*02 confers a protective influence. Indeed, it is intriguing that most of the SNPs are associated with CD4+T cell regulation. Contemporary screening techniques have further evolved with even greater refinement in genetic element identification conferring risk of MS by virtue of the utilization of the powerful technique of whole exome sequencing (WES), that provides important perspective on differential gene expression mechanisms. Finally, the GWAS discovery associated with MS of the CYP27B1 gene, has particular relevance to an epigenetic disorder such as MS, given that this gene encodes for the critical enzyme that serves to transform the 25-OH-D form to the 1,25-OH-D active form of vitamin D. With respect to the pathophysiologic relevance of the hnRNPA1 variants, these appear to alter neuronal function, and ultimately culminate in neurodegeneration, in conjunction with serving a role in the important regulation of viral synthesis (another highly conspicuous epigenetic factor with special relevance to MS and related disorders). As the authors rightly point out, to date the somatic mutations in question and under investigation have only been elucidated from PBMCs of MS patients. This work, if confirmed, may have special and critical relevance to the highly enigmatic phenomenology of what determine the transition from a relapsing to a progressive form of MS (or a progressive form from the inception without an antecedent relapsing and adaptive immune phase of the disorder), and such studies might shed light on the pathobiological underpinnings of axonal and neuronal degeneration; the histopathological signatures of what we currently believe to constitute the signature of irreversible (at least so far) neurologic disability with corresponding loss of functional capabilities. This work, contributes to our deeper understanding of the potential mechanisms of disease that figure prominently in influencing progression and neurodegeneration for MS in particular, but may also shed light on those mechanisms that underly neurodegenerative disorders in general. The authors are to be congratulated for this fine contribution to the literature.", "responses": [ { "c_id": "977", "date": "11 Sep 2014", "name": "Michael Levin", "role": "Author Response", "response": "We appreciate Professor Frohman’s insightful commentary about our work. We concur that to date, we have only found somatic nucleotide variants (SNVs) in PBMCs of MS patients. Further studies are warranted about the contribution of SNVs to axonal and neuronal degeneration in the CNS of MS patients. This is addressed in the last two paragraphs of the discussion." } ] } ]	1	https://f1000research.com/articles/3-132
https://f1000research.com/articles/3-225/v1	18 Sep 14	{ "type": "Opinion Article", "title": "Chile’s dilemma: how to reinsert scientists trained abroad", "authors": [ "Alexia Nunez-Parra", "Maria-Paz Ramos" ], "abstract": "Chile is recognized worldwide as an emergent economy, with a great power in natural resource exploitation. Nonetheless, despite being one of the most developed countries in Latin America, Chile imports most of the knowledge and technology necessary to drive innovation in the country. The tight budget that the Chilean government assigned to research and development and the absence of a long-term scientific agenda contributed to a limited supply of scientists over the years. In an effort to reverse this scenario, Chile has created several fellowships, such as the Becas Chile Program (BCP) to encourage new generations to pursue graduate studies to ultimately advance research and development in situ. More than 6000 fellows are now being trained abroad, accumulating an incredible potential to transform the Chilean scientific environment as we know it. Chile now faces a greater challenge: it has to offer infrastructure and job openings to the highly skilled professionals in whom it invested. Unfortunately no clear public policies to address this situation have been developed, partially due to the lack of a dedicated institution, such as a Ministry for Science and Technology which could focalize the necessary efforts to promote such policies. Therefore, in the meantime, Chilean scientist have been motivated to create different organizations, such as, Mas Ciencia para Chile and Nexos Chile-USA, to promote constructive discussion of the policies that could be implemented to improve the Chilean scientific situation. We hope that these and other organizations have a real impact on the generation of scientific guidelines that will finally contribute to the development of the country.", "keywords": [ "Chile is catalogued worldwide as one of the most stable countries in the Latin-American region. It is seen as an example of a growing economy", "experiencing an average gross domestic product (GDP) growth rate of 5.3% in the last four years (World Development Indicators", "World Bank data). This economic stability continuously attracts several international corporations that settle and open new branches in its territory. Chile is referred to as a flourishing nation with an intrinsic desire to innovate and reach international technological standards. Moreover", "in 2010 Chile became the first South American country to join the Organization for Economic Cooperation and Development (OECD)", "recognizing Chile’s prosperous economy and strong democratic union." ], "content": "Commentary\n\nChile is catalogued worldwide as one of the most stable countries in the Latin-American region. It is seen as an example of a growing economy, experiencing an average gross domestic product (GDP) growth rate of 5.3% in the last four years (World Development Indicators, World Bank data). This economic stability continuously attracts several international corporations that settle and open new branches in its territory. Chile is referred to as a flourishing nation with an intrinsic desire to innovate and reach international technological standards. Moreover, in 2010 Chile became the first South American country to join the Organization for Economic Cooperation and Development (OECD), recognizing Chile’s prosperous economy and strong democratic union.\n\nThe Chilean economy is fundamentally sustained by natural resources, such as copper extraction, as well as fruit production and exportation. Therefore, the next logical step would be to invest in novel technology to expand the already established industries, and furthermore, define new strategies to generate sustainable growth. Paradoxically, despite its economic power, only 0.42% of the GDP is assigned to Research and Development (R&D) in Chile (Research and development expenditure, World Bank data). Moreover, the funds for the formation of advance human capital have been historically low. As an attempt to reverse that situation, Chile created the Republic President Fellowship program in 1981. The tight budget, however, considered only two years of specialization, excluding the possibility of pursuing longer programs, such as a PhD. In addition to the governmental program, international organizations, such as Fulbright, collaborated with the Chilean National Commission for Science and Technology (CONICYT) to grant fellowships to Chilean students to study abroad. This program, known as the Equal Opportunities Fellowship, was discontinued due to the great monetary investment that it involved.\n\nIn an effort to promote the development of the sciences, Chile set a long term goal to join the knowledge society in which knowledge is generated and shared to ultimately improve the human condition through innovation and technology. As a first approach, and in order to inform the community about the importance of science, CONICYT created several programs, such as the Explora projects, to teach middle school children science in a didactic way, or La Ciencia nos cambia la Vida, mini documentaries about scientific developments created in Chile that impact the quality of life of its own population. Moreover, as part of the plan to reach this ambitious goal, in 2008 the Chilean government created the Becas Chile Program (BCP). This fellowship, under the direction of CONICYT, aims to increase the opportunities of continuing education in foreign countries, coordinates the national fellowship program, and enhance international cooperation. Seizing the opportunity, Chilean students and professionals applied for the fellowships and massively enrolled in graduate programs (masters and PhDs) and postdoctoral programs in several countries worldwide. The number of fellows jumped from less than 200 annually before the creation of the BCP, to more than 1200 within the first year of the program implementation (Statistics indicators, Becas CONICYT International). After six years of operation, 6,042 fellowships have been granted in order to pursue masters and doctorates abroad (Statistic indicators, Becas Chile).\n\nWith the implementation of BCP, an important and historical advancement in the formation of human capital has been made. Chile will have for the first time a number of highly specialized professionals to advance science, technology and innovation. However, this explosive increase in the number of available fellowships carried enormous consequences. The lack of an institution controlling the necessary policies (such as a dedicated ministry) focused on reintegrating the highly skilled professionals trained abroad back into the Chilean academic and industrial system, has become evident. This problem was already detected and presented in the conclusions of the revision of the BCP performed by the OECD in partnership with the World Bank in 20101. In this review, they state, “more purposeful efforts will be needed to maximize the re-integration of BCP graduates, especially in several fields where the country has limited scale and scope”. Including scientists studying abroad by other means (i.e. supported by international grants or academic programs), we estimate that the number of scientists that will have to be reinserted in Chile adds several hundreds more per year to the already 6,000+ BCP fellows. The problem gets aggravated by the fact that the fellowships stipulate that the recipients have to return to Chile after the fellowships end for twice the number of years they were studying abroad. If they do not return, they have to pay back the investment granted for their education. It is expected that Chile would reap the rewards of investing in human capital training and prevent brain drain. Nevertheless, the country is unfortunately not fully prepared to benefit from all these professionals as there are not enough jobs available nor the infrastructure required to support them.\n\nIn order to improve that situation, some efforts have been taken to offer opportunities and take advantage of the new experts. CONICYT has recently created fellowships and grants to attract professionals that are residing abroad, but the number of fellowships available is still limited, reaching no more than 25 per year (Attraction and Insertion of Advanced Human Capital Program, CONICYT).\n\nThe weaknesses of the scientific system in Chile that are leading to sparse permanent job openings have been discussed by several institutions and organizations. Some of the most recurrent ones mentioned are Chile’s lack of governance and scientific support and the reduced involvement of the private sector in the development of science and technology2. The planning, development and implementation of public policies in science occurs in different ministries and institutions to the detriment of well-planned long-term policies and scientific agenda (Final report 2013, National Presidential Commission). Without clear guidelines, it seems difficult to create a stable scientific system fed by basic and applied research that could lead to economic growth in the country. This is in contrast to most other OECD countries that contain a Ministry of Science and Technology or similar institution that helps to converge efforts for advancement in science, technology and innovation. Moreover, even though the budget assigned for research and development has been doubled since 2009, reaching 1.037 billion dollars in 2013 (CONICYT Annual Report 2013), it is still considered very limited considering that it has been maintained at around 0.4% GDP for the last five years. This situation contrasts with the percentage that others countries in the region designate to science, for instance Argentina and Brazil contribute approximately 1% of their GDP. In addition, as opposed to what occurs in more developed countries, the interest of the private sector in investing into scientific research is minimal. Chile does not have a system that provides incentives to private fundraising foundations, nor companies to generate technology and innovation in situ.\n\nNevertheless, despite the lack of considerable funding, the quality of science that is produced in Chile is one of the best in the region, being the country with the highest ranking in terms of number of ISI publications per hundred million dollars invested and per number of inhabitants (CONICYT and RYCIT). The high quality science performed in Chile and the fact that thousands of highly prepared scientists will populate the country in the next years creates incredible potential for the prospect of Chilean science. The current scenario is ideal for Chile to make the jump forward by taking advantage of the situation and positioning Chilean science as one of the top in the world. Today is the moment to increase the scientific budget and define a clear strategy for the reinsertion of the thousands of professionals that will go back to Chile to implement the state-of-the-art technologies and knowledge acquired abroad. At the same time, new buildings and laboratory space specially designed to meet the needs of the researchers are required and badly needed.\n\nThe lack of an institution in charge of the scientific development will hardly change in the short-term. President Michelle Bachelet has not disclosed a clear agenda to promote public policies in science. While the Senate has sent a request to create the Ministry for Science and Technology, the necessary laws may take several years to be promoted, especially since the efforts of the current administration are mainly focused in the popular concern of transforming the educational and tax systems (Program of Government, Chilean Government). Even though a general feeling regarding the importance of the Chilean scientist abroad has started to grow, researchers observe with concern the lack of policy implementations needed for scientific development in Chile and the real possibilities of finding interesting opportunities back home.\n\nIn such a scenario, a group of scientists in Chile started a social movement to generate a productive discussion about the importance of science and technology in the development of a country. The organization they created at the end of 2010, called Mas Ciencia para Chile, is today a foundation that aims to promote the debate about the importance of developing scientific policies in Chile. In parallel and with a similar perspective, Chilean researchers residing abroad have realized the necessity of contributing in the meantime, and eventually in parallel with the appropriate authorities, to promote the reinsertion of their newly advance trained group into the country. As an attempt to discuss such strategies and gather the large number of scientists working and studying in the United States, Nexos Chile-USA was founded in 2010 (www.nexoschileusa.org). Nexos aims to communicate and develop collaborations between its members, and more importantly, to promote interaction between Chilean scientists in the USA and their peers in Chile. In addition, Nexos Chile-USA has become a platform to inform its members about different paths for the reinsertion after their training abroad, and to offer options for alternative careers outside academia. Nexos has created multiple tools to communicate with its community: from monthly newsletters about grant or job opportunities, interviews with prestigious scientists who give advice on how to build a scientific career, to documents guiding PhD applications and life in the U.S.\n\nPossibly, the most valuable instrument that Nexos Chile-USA has implemented to pursue its aims is its annual meeting, which in close collaboration with the Embassy of Chile in Washington D.C., gathers around 150 scientists from all around the country. This annual meeting is an opportunity to listen to scientific talks of great value from other Chilean scientists and workshops aimed at facilitating professional careers, as well as a tool to discuss scientific polices with relevant players of main funding agencies in Chile. Finalizing this year’s meeting at in October, the organizing committee plans to elaborate and disseminate a document with the predominant topics discussed during the meeting and the round table: “Chilean Science: An attractive pole for international collaboration?” including the perspective the community abroad has about the situation in Chile. With such a document, Nexos Chile-USA aims to actively participate in the discussion about the necessary political and scientific infrastructure that will help the advance of science in Chile, in addition to stimulating other organizations and institutions to provide their own novel ideas about such an important topic.\n\nThe importance of organizing the Chilean scientific community abroad has been considered in, and spread to, multiple countries, where professionals have created organizations similar to Nexos Chile-USA (including REDICEC in Canada, Red INVECA in Germany, REUK and RedICE in the UK, Ech Francia in France, RedInche in Spain, CREGA in Australia, RIECH and Becpass, among others). These different entities have met multiple times to discuss the necessity of contributing to the discussion of public policies to promote science in Chile and to facilitate a better understanding between the government and the scientific community. We hope that in the near future Nexos Chile-USA, together with these other organizations, will contribute to the development of the country.", "appendix": "Author contributions\n\n\n\nANP and MPR both helped to design and write the manuscript as well as compile the relevant information and statistical data. Both authors read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nOECD and the International Bank Reconstruction and Development/The World Bank. Reviews of National Policies for Education Chile’s International Scholarship Programme. OECD publisher 2010. Reference Source\n\nAstudillo Besnier P: Chile needs better science governance and support. Nature. 2014; 511(7510): 385. PubMed Abstract \| Publisher Full Text" }	[ { "id": "6175", "date": "22 Sep 2014", "name": "Jorge Babul", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis Commentary addresses an important problem for the scientific development of Chile: the training of highly specialized professionals in foreign countries and their reintegration to the Chilean academic and industrial world. The authors describe the Chilean efforts to promote the advancement of science, the weaknesses of the scholarship system, the lack of governance and support, and how scientists in Chile and abroad are getting organized in order to develop scientific policies to solve these problems.There are a few minor revisions that should be made:Number of fellowships. It should include separately masters, PhDs and postdoctoral fellowships (only the total number, 6,042, is given). \"[...] partnership with the World Bank in 20101.\" Correct year 2010? \"The weaknesses of the scientific system in Chile that are leading to sparse permanent job openings have been discussed by several institutions and organizations.\" Mention should be given to the main ones. \"[...] development of science and technology2.\" Delete 2. \"[...] at around 0.4% GDP for the last five years.\" The number of years should be revised (I would say for the last 15 years). Before Chile joined the OECD, the budget was 0.7% GDP; afterwards it had to be recalculated according to OECD rules, giving 0.4%. \"Chile does not have a system that provides incentives to private fundraising foundations, nor companies to generate technology and innovation in situ.\" It has an incentive system for cooperation between industries and research institutions, the so called “I+D Law”. \"[...] positioning Chilean science as one of the top in the world.\" This is overstated. \"[...] for the reinsertion of the thousands of professionals that will go back to Chile.\" Although this commentary is about reinsertion of professionals that were trained abroad, the number of PhD students that are trained through the national program of scholarships should also be considered; they are part of the problem. Compare the number of PhD scholarships/students with national and international scholarships, for example. \"[...] this year’s meeting at in October.\" Delete at. \"[...] will help the advance of science in Chile.\" advancement?", "responses": [] }, { "id": "6176", "date": "22 Sep 2014", "name": "Gabriel Leon", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis Opinion Article addresses an important issue that needs to be answered by the Chilean Government: how to reinsert the young scientists trained abroad. In the past years, the Chilean Government has developed international training programs but little has been done in order to generate infrastructure and job openings for those highly trained scientists. This article describes how the scientists are organizing and pushing in order to generate policies that address this problem and others related to the Chilean scientific development, as the dependence on the exploitation of natural resources and how to transform the economy to a knowledge-based one. Minor revisions:\"...with a great power in natural resource exploitation\". Change power to emphasis. \"Finalizing this year’s meeting at in October\", delete \"at\". Even when this Opinion Article is related to the problem of the reinsertion of scientists trained abroad, is necessary to include in this analysis the number of scientist that were trained in Chile. They are additive to this scenario. \"ANP and MPR both helped to design and write the manuscript...\" change to \"ANP and MPR wrote and edited the manuscript...\"", "responses": [] } ]	1	https://f1000research.com/articles/3-225
https://f1000research.com/articles/2-243/v2	15 Nov 13	{ "type": "Research Article", "title": "The electrostatic profile of consecutive Cβ atoms applied to protein structure quality assessment", "authors": [ "Sandeep Chakraborty", "Ravindra Venkatramani", "Basuthkar J. Rao", "Bjarni Asgeirsson", "Abhaya M. Dandekar", "Ravindra Venkatramani", "Basuthkar J. Rao", "Bjarni Asgeirsson", "Abhaya M. Dandekar" ], "abstract": "The structure of a protein provides insight into its physiological interactions with other components of the cellular soup. Methods that predict putative structures from sequences typically yield multiple, closely-ranked possibilities. A critical component in the process is the model quality assessing program (MQAP), which selects the best candidate from this pool of structures. Here, we present a novel MQAP based on the physical properties of sidechain atoms. We propose a method for assessing the quality of protein structures based on the electrostatic potential difference (EPD) of Cβ atoms in consecutive residues. We demonstrate that the EPDs of Cβ atoms on consecutive residues provide unique signatures of the amino acid types. The EPD of Cβ atoms are learnt from a set of 1000 non-homologous protein structures with a resolution cuto of 1.6 Å obtained from the PISCES database. Based on the Boltzmann hypothesis that lower energy conformations are proportionately sampled more, and on Annsen's thermodynamic hypothesis that the native structure of a protein is the minimum free energy state, we hypothesize that the deviation of observed EPD values from the mean values obtained in the learning phase is minimized in the native structure. We achieved an average specificity of 0.91, 0.94 and 0.93 on hg_structal, 4state_reduced and ig_structal decoy sets, respectively, taken from the Decoys `R' Us database. The source code and manual is made available at https://github.com/sanchak/mqap and permanently available on 10.5281/zenodo.7134.", "keywords": [ "Computational biology", "protein structure prediction", "Model quality assessment programs", "Boltzmann distribution", "Decoys `R' Us", "Annsen's thermodynamic hypothesis", "Finite difference PoissonBoltzmann (FDPB)", "APBS", "statistical potentials", "protein sidechain", "decoy sets", "template based modeling", "ab initio protein structure prediction" ], "content": "Introduction\n\nThe challenge of deriving the native structure of a protein from its sequence has intrigued researchers for decades1. Methods that predict putative structures from sequences are based either on features from databases of known structures (template-based methods)2–4 or use first principles of atomic interactions (ab initio or de novo methods)5–7. Typically, these methods yield multiple, closely-ranked possibilities. Model quality assessment programs (MQAP) that validate accuracy of these predicted structures are used to select the best candidate from the set of predicted structures.\n\nMQAPs can be classified as energy, consensus or knowledge based. Two major sources of errors in energy based methods used for refining or discriminating protein structures are inaccuracies in the force field due to the inherent approximations in equations that model multi-atomic configurations, and inadequate sampling of the conformational space8–12. Consensus based methods are based on the principle that structural features that are frequently observed in a population of structures are more likely to be present in the native structure13–16. These clustering methods outperform other MQAP methods14 and are \"very useful for structural meta-predictors17\". However, they are prone to be computationally intensive due structure-to-structure comparison of all models16, and are of limited use when the number of possible structures is small18. Knowledge based methods proceed by deriving an empirical potential (also known as statistical potential) from the frequency of residue contacts in the known structures of native proteins19,20. For a system in thermodynamic equilibrium, statistical physics hypothesizes that the accessible states are populated with a frequency which depends on the free energy of the state and is given by the Boltzmann distribution. The Boltzmann hypothesis states that if the database of known native protein structures is assumed to be a statistical system in thermodynamic equilibrium, specific structural features would be populated based on the free energy of the protein conformational state. Applying a converse logic, Sippl reasoned that the frequencies of occurrence of structural features such as interatomic distances in the database of known protein structures could be used to assign a free energy (potential of mean force) for a given protein conformation21,22. Furthermore, this statistical potential can be used to discriminate the native structure23–27. The proper characterization of the reference state is a critical aspect in applying statistical potentials23. In spite of their popularity, the application of such empirical energy functions to predict and assess protein structures are vigorously debated28,29. Many MQAP programs perform better when multiple statistical metrics are combined30–33. The paramount importance of obtaining high quality protein structures from sequences using in silico methods can be estimated by the effort invested by researchers every two years34 to evaluate both structure prediction tools35 and MQAPs17,34,36.\n\nHere, we propose a novel statistical potential to assess the quality of protein structures based on the electrostatic potential difference (EPD) of Cβ atoms in consecutive residues - EPD profile of sidechain atoms used in assessment of protein structures (ESCAPIST). Previously, we have established that the EPD is conserved in cognate pairs of active site residues in proteins with the same function37–40. The ability of finite difference methods to quickly obtain consistent electrostatic properties from peptide structures provides an invaluable tool for investigating other innate properties of protein structures41. We plot the EPD profiles for different atom types (Cα atoms, Cβ atoms and the C-N bond) in consecutive residues from a set of non-homologous protein structures obtained from the PISCES database (http://dunbrack.fccc.edu/PISCES.php)42. We proceed to show that the EPD between Cβ atoms in consecutive residues can be used to generate a scoring function that assesses the quality of protein structures. This EPD scoring function is then applied to standard decoy sets from the Decoys ‘R’ Us database (http://dd.compbio.washington.edu) to establish the validity of our method43.\n\n\nResults\n\nTo extract feature values we chose a set of 1000 proteins from the PISCES database with percentage identity cutoff of 20%, resolution cutoff of 1.6 Å and a R-factor cutoff of 0.25 (SI Table 1).\n\nWhile these pairs have for a low standard deviation (SD) like all other pairs, the absolute value of their mean is different (higher) than any pair that does not include a proline. This also highlights the unique nature of proline in protein structures.\n\nAdaptive Poisson-Boltzmann Solve (APBS) writes out the electrostatic potential in dimensionless units of kT/e where k is Boltzmann’s constant, T is the temperature in K and e is the charge of an electron. The units of EPD are same as that of the electrostatic potential. The EPD of the C-N peptide bond has a Gaussian distribution with mean = 420 EPD units and SD = 55 EPD units (Figure 1). In the probability distribution for four pairs of amino acids the mean of all pairs of amino acids are the same (Figure 1a). Figure 1b shows the scatter plot for the mean and standard deviation (SD). Thus, the amino acids are indistinguishable using the profile of the EPD of the C-N peptide bond across all protein structures since they have identical mean values and a large variance (SD=~50).\n\nAA: Alanine/Alanine, AC: Alanine/Cysteine, HS: Histidine/Serine and DF: Aspartic-acid/Phenylalanine. (a) Probability distribution for four pairs of amino acids. (b) Scatter plot for all pairs of amino acids. It can be seen that the mean and SD for all pairs of amino acids are the same. Further, the variance is large (SD=~50), indicating that this feature is not tightly constrained in peptide structures.\n\nThe probability distribution for four pairs of amino acids for the EPD between the Cα atoms of consecutive residues (Figure 2a) have means that are slightly more varied than those for the C-N bond (Figure 1a). In the scatter plot for the mean and SD of all pairs (Figure 2b) the outliers are pairs that include proline, which have a higher mean, although the magnitude of SD is the same (Table 1).\n\nA: Alanine/Alanine, AC: Alanine/Cysteine, HS: Histidine/Serine, DF: Aspartic-acid/Phenylalanine. (a) Probability distribution for four pairs of amino acids. (b) Scatter plot for all pairs of amino acids. It is seen that pairs of amino acids which include proline have a higher mean, although the magnitude of SD is the same.\n\nIn contrast to the results described above, the EPD between the Cβ atoms in consecutive residues in the peptide structure can be used to discriminate different amino acid pairs in the protein structure. The mean EPD of all amino acid pairs are much more varied (Figure 3a). These pairs do not include glycine, which lacks a sidechain. In the scatter plot for the mean and SD, the outliers are pairs that include cysteine (Figure 3b), which have a much higher SD (=~90) as compared to other pairs (SD=~35) (Table 2), and thus cannot be used for discriminatory purposes.\n\nAA: Alanine/Alanine, AD: Alanine/Aspartic-acid, AE: Alanine/Glutamic-acid, DF: Aspartic-acid/Phenylalanine, DY - Aspartic-acid/Tyrosine, HT: Histidine/Threonine, HS: Histidine/Serine. (a) Probability distribution for seven pairs of amino acids. (b) Scatter plot for all pairs of amino acids. The pairs which include cysteine have a high standard deviation. It is seen that the mean is much more varied than the electrostatic potential difference (EPD) for Cα and the C-N peptide bond.\n\nThese pairs have a random values for the mean and a high standard deviation (SD), with the exception of the pair ‘CC’ (not the disulfide bond) which has a low mean value and SD. Consequently, these values can not discriminate between pairs of amino acids.\n\nThese values are used as a discriminator when choosing the native structure from a set of possible candidates (Table 3). To establish the non-triviality of these values, we also show that the variance of the EPD between these pairs increases with increasing sequence distance. Thus, the EPD between the pairs ‘DF’ and ‘HS’ has lesser correlation as the sequence distance between them increases (sample size for each sequence distance is > 30) (Figure 4). The SD for distance 1 (i.e. consecutive residues) is 29.8 EPD units and 31.8 EPD units for ‘DF’ and ‘HS’, respectively - and rises to around 60 EPD units with increasing sequence distance.\n\nThese pairs are used for discriminating predicted structures in order to obtain the native structure. The complete set is available at https://github.com/sanchak/mqap.\n\nEach sequence distance has at least 30 sample points. DF: Aspartic-acid/Phenylalanine, HS: Histidine/Serine. As expected, there is lesser correlation in the EPD values between the shown amino acid pairs ‘DF’ and ‘HS’ as the sequence distance between the residues increases. The SD for distance 1 (i.e. consecutive residues) is 29.8 EPD units and 31.8 EPD units for ‘DF’ and ‘HS’, respectively - and rises to around 60 EPD units with increasing sequence distance.\n\nWe obtained the score (PDScore) of any given protein structure by comparing the electrostatics of the Cβ atoms based on Table 3. To benchmark model quality assessment programs, we used decoy sets from the Decoys ‘R’ Us database43. We detail our results from some of these decoy sets. Each set has several structures that are supposed to be ranked worse than the native structure.\n\nThe misfold decoy set has ~20 protein structures, each of which has a correct and an incorrect structure specified (three structures have two incorrect structures: we randomly chose the first)44. The PDScore of these proteins were computed (Table 4). Barring three structures (PDBids: 1CBH, 1FDX and 2SSI), the PDScore of the incorrect structure is higher than that of the correct structures.\n\nThis decoy set has ~20 protein structures - each of which has a correct and an incorrect structure specified. The PDBs are sorted based on the number of residues in the structure (NRes). Three of the structures (1CBH, 1FDX and 2SSI) have a lower PDScore for the incorrect structure.\n\nThe hg_structal set has about ~30 proteins. Each protein has 30 structures (including the native structure). Table 5A shows specificity obtained for structures in this decoy set. The average specificity obtained for this decoy set is 0.91 (Table 5A). The decoy set 4state_reduced has ~600 structures for each of the seven proteins. We obtain an average specificity of 0.94 for this decoy set (Table 5B). Similarly, for the ig_structal decoy set we obtain a specificity of 0.93 (Supplementary Table 1).\n\nThe PDBs are sorted based on specificity. (A) The hg_structal decoy set has ~30 protein structures - each of which has 30 structures. The average specificity obtained for the set is 0.91. (B) The 4state_reduced decoy set has 7 protein structures - each of which has ~600 structures. The average specificity obtained for the set is 0.94. (C) The fisa set has 4 protein structures - each of which has 500 structures. The electrostatic disciminator has low specificities in this case. We have previously demnostrated that this decoy set can be discriminated by a distance based criterion. It consists of physically nonviable structures, thus rendering an electrostatic analysis meaningless. NRes = number of residues, NStructures = number of structures in the decoy set.\n\n\nDiscussion\n\nThe functional characterization of a protein from its sequence using in silico methods based on the ‘sequence to structure to function’ paradigm is contingent upon the availability of its 3D-structure. The rapidly developing field of next generation sequencing has exacerbated the bottleneck of obtaining structural data using crystallization techniques45. This ever-widening gap has been filled by methods that predict structures from sequences46, based either on features from databases of known structures2–4 or from first principles of atomic interactions5,6.\n\nThe various sources of error in protein structure prediction have been previously discussed in detail47. An incorrect model of a protein structure will result in an inaccurate analysis of its properties48. For example, continuum models49 that compute potential differences and pKa values from charge interactions in proteins50 are sensitive to the spatial arrangement of the atoms in the structure. Accurate structural information is indispensable for in silico methods that extract the electrostatic profile of atoms in the peptide structure41,51, and for other methods widely used in pharmacology for drug discovery52. Model quality assessment programs (MQAP) that validate the accuracy of predicted structures are thus a critical aspect in the process of modeling a protein structure from its sequence. MQAPs can be classified as energy8–12, consensus13–16 or knowledge based (statistical potential)21–27. The state of the art methods for predicting structures35 and MQAPs17,34,36 are evaluated by researchers every two years.\n\nPreviously, we hypothesized and demonstrated that the electrostatic potential difference (EPD) in cognate pairs in the active site are conserved in proteins with the same functionality37,40,53, even when evolution has converged to the same catalytic from completely different sequences54. This similarity is observed in structures solved independently over many years and establishes the reliability of the APBS and PDB2PQR implementations41,55. We focused on unraveling other electrostatic features that are innate to protein structures. Here, we first demonstrate that the EPD between the C-N peptide bond and the Cα atoms of consecutive residues are independent of the amino acid type. This is expected, since the distance between these atoms are almost invariant across all structures. The EPD of the C-N bond has a high variance, implying that the backbone accommodates relatively large variations while seeking energetically minimized structures.\n\nThe true source of the chemical and structural diversity in protein structures is the side chain atoms. Every amino acid, except glycine, has a Cβ atom that hosts a unique moiety of atoms. Although the reactive groups are different for amino acids, we show that this difference is encapsulated in the backbone Cβ atoms. We first show that different pairs of amino acids have significantly different mean EPD values in side chain Cβ atoms (Figure 3), unlike the EPD of the C-N peptide bond (Figure 1) or the EPD between consecutive Cα atoms (Figure 2). Further, the variance is much less than in the EPD of the C-N bond. These facts suggested that the EPD between Cβ atoms of consecutive residues in the peptide structure might act as a discriminator. Our hypothesis is based on the insightful Boltzmann law that lower energy conformations are disproportionately sampled, on the thermodynamic hypothesis56 that the native structure has minimal energy, and the hypothesis that statistical derived features in known protein structures have a Gaussian distribution21. We apply our discriminator to standard decoy sets from the Decoys ‘R’ Us database to establish the validity of the method43.\n\nOur work also highlights the unique properties of proline in the protein structure57. This is evident from the different magnitude of EPD in consecutive Cα atoms involving proline (Table 1). Another noteworthy aspect is the large variation in EPD in consecutive Cβ atoms involving cysteine (Table 2), demonstrating the unique role cysteine plays in providing flexibility to protein structures, a critical element in the evolution of complex organisms58. The discrimination of Cβ atoms also provides a uniform basis for methods that require a single-atom representation of a residue. Such methods depend on a correct parameterization of the reactive atoms37, a task further complicated by amino acids such as histidine which has two reactive atoms. For example, the EPD between the negatively charged E and D with respect to the aromatic phenylalanine is -108 and -93 EPD units, in spite of the difference in their reactive atom. Similarly, the EPD between alanine and the three aromatic amino acids (F, W and Y) are -67, -66 and -63 EPD units respectively.\n\nWe achieved an average specificity of 0.91, 0.94 and 0.93 on hg_structal, 4state_reduced and ig_structal decoy sets, respectively, taken from the Decoys ‘R’ Us database. We have previously demonstrated that the fisa decoy set can be discriminated by a distance based discriminator59. ESCAPIST does not discriminate the native structure in this decoy set (Table 5C). The physical implication of ESCAPIST results on the fisa decoy set, which has significant RMSD for backbone Cα atoms, needs further elaboration. The input to a finite difference Poisson-Boltzmann (FDPB) analysis is a charge distribution that might be unfeasible due to energy functions other than electrostatics. For example, van der Waals force or the elastic bond length force components might prevent two atoms from being in close proximity. However, if such a physically impossible configuration were presented to a FDPB-based analysis tool, such as APBS41, it would still generate an electrostatic potential landscape. Inferences based on this potential landscape would be incorrect due to its physical non-viability. Thus, before invoking the EPD constraints specified here, it is imperative that other spatial constraints that are rarely violated in structures are checked. Possibly for this same reason, MQAPs that combine many features in their scoring functions are superior. Moreover, it should be kept in mind that decoy sets, like most benchmarking sets, are prone to biases60 and possible errors31. In fact, the fisa decoy set has been shown to violate the van der Waals term60. To summarize, we present a novel discriminating feature in protein structures based on the electrostatic properties of the side chain atoms. We validated this discrimination in several decoy sets taken from the Decoys ‘R’ Us database, and achieved high specificities in most decoy sets.\n\n\nMethods\n\nOur proposed method has two phases. In the learning phase, we process multiple structures to extract the feature values - mean values of electrostatic potential difference (EPD) for each amino acid pair. These feature values are applied on query proteins to obtain a score (PDscore) that indicates the deviation of the feature values in the given structure from the ‘ideal’ values. Thus, a lower PDscore indicates a better candidate.\n\nAlgorithm 1 shows the procedure LearnFeatures() that extracts the feature values from a set of proteins ΦLearningPhaseproteins (Equation 1). We ignore the first x=IgnoreNTerm and last y=IgnoreCTerm pairs of residues in the protein structure to exclude the terminals. For every consecutive pair of residues in the structure, we calculate the EPD (see below for method) between two specified atoms (atomP and atomQ). Both atomP and atomQ are set to Cβ to obtain EPD values for Cβ atoms, while we set atomP to ‘C’ and atomQ to ‘N’ in order to obtain the C-N peptide bond EPD values. The mean (Mean learnt value - MLV) and standard deviation (SD) are computed for each amino acid type pair (AAType) in protein (Equation 2), and the mean computed for the globals set of proteins (MLV(AATypex, AATypey)) for each pair of amino acid types (Equation 3). Pairs whose EPD have a SD greater than a threshold value (sdThresh, 50 by default) are ignored. Means for all significant pairs (ϕpairMean) are noted to a file, which is the input to the quality assessment phase. The EPD between a pair of amino acid is order-independent - for example, the EPD statistics for the pair ‘AC’ (alanine-cysteine) includes the EPD of both ‘AC’ and ‘CA’ (with the sign reversed).\n\n\n\n\n\n\n\nAlgorithm 2 shows the function AssessEPDQuality() that generates the PDscore for a given protein from the template file generated by the learning phase. The set of proteins ΦAssessment Phaseproteins consists of the native structure P1 and N-1 decoys structures (Equation 4). Once again, barring x=IgnoreNTerm and y=IgnoreCTerm number of residues from the N and C terminals, the pairwise EPD for consecutive residues are computed. The absolute value of the difference of these values from their corresponding means, if they exist, in the template file is added to generate the absolute score (Equation 5). This score is normalized with the number of residues that have been compared to obtain the final PDscore. In summary, the PDscore encapsulates the average distance of the EPD for the given atom pairs (it may be Cβ, Cα or the C-N bond) of consecutive residues from their mean values. We hypothesize that in the native or a near native structure, the PDscore will be minimized for the EPD of Cβ atoms of consecutive residues, i.e. given a set of proteins Φproteins consists of the native structure P1 and N-1 decoys structures, P1 will have the minimum PDscore (Equation 6).\n\n\n\n\n\n\n\nThe top level procedure ESCAPIST() is shown in Algorithm 3. It invokes the function LearnFeatures() once, and applies the learnt values to assess the quality of structures based on the feature values obtained.\n\nAdaptive Poisson-Boltzmann Solver41 (APBS) and the PDB2PQR package55 package was used to calculate the potential difference between the reactive atoms of the corresponding proteins. The APBS parameters are set as follows - solute dielectric: 2, solvent dielectric: 78, solvent probe radius: 1.4 Å, Temperature: 298 K and 0 ionic strength. APBS writes out the electrostatic potential in dimensionless units of kT/e where k is Boltzmann’s constant, T is the temperature in K and e is the charge of an electron.\n\nInput: ϕproteins = {P1 ··· PM} : M Proteins in the learning set\n\nInput: IgnoreNTerm: Ignore this number of residues in the N Terminal\n\nInput: IgnoreCTerm: Ignore this number of residues in the C Terminal\n\nInput: atomP: Atom type in first residue\n\nInput: atomQ: Atom type in second residue\n\nInput: sdThresh: Threshfold for standard deviation of the EPD\n\nOutput: ϕpairMean = {meanPDC1 ··· meanPDCK}: Mean values of EPD between specified atoms\n\nof successive residues, there being K such significant pairs\n\nbegin\n\n/K pairs of amino acid type (sorted: AC and CA are equivalent)/\n\n/Each set is initialize to be the null set/\n\nϕpair = {ϕ1PDC ··· ϕKPDC} :\n\nforeach Pi in ϕproteins do\n\nN = NumberOfResidues(Pi);\n\nfor p ← IgnoreNTerm to (N − IgnoreCTerm) do\n\nq = p + 1 ;\n\n/* Amino acid pairs are order independent /\n\nResiduePairTypeString = ResidueTypeString(p) + ResidueTypeString(q);\n\nResiduePairTypeStringSorted = Sort(ResiduePairTypeString;\n\n/ Reverse sign of potential difference accordingly /\n\nmultfactor = 1 ;\n\nif (ResiduePairTypeStringSorted != ResiduePairTypeString) then\n\nmultfactor = -1 ;\n\nend\n\nPD = PotentialDifference(p, q, atomP, atomQ) multfactor ;\n\n/* Let the amino acid pair be the kth in the set ϕpair /\n\nInsertInSet(PD, ϕkPDC);\n\nend\n\nend\n\n/ Compute Mean and SD of each set - ignore pairs with SD greater than sdThresh/\n\nϕpairMean = ∅;\n\nforeach ϕipair in ϕpair do\n\n(Meani, SDi) = MeanAndSD(ϕipair);\n\nif (SDi > sdThresh) then\n\nAdd(Meani, ϕpairMean);\n\nend\n\nend\n\nreturn (ϕpairMean);\n\nend\n\nInput: P1 : Protein under consideration\n\nInput: IgnoreNTerm: Ignore this number of residues in the N Terminal\n\nInput: IgnoreCTerm: Ignore this number of residues in the C Terminal\n\nInput: atomP: Atom type in first residue\n\nInput: atomQ: Atom type in second residue\n\nInput: ϕpairMean = {meanPDC1 ··· meanPDCM}: Mean values of EPD between specified atoms of\n\nsuccessive residues\n\nOutput: PDscore: Score indicating the normalized distance of the observed values from the (mean)\n\nlearnt values from native structures\n\nbegin\n\nPDscore = 0 ; NumberCompared = 0 ; N = NumberOfResidues(P1);\n\nfor p ← IgnoreN T erm to (N − IgnoreCT erm) do\n\nq = p + 1 ;\n\n/ Amino acid pairs are order independent /\n\nResiduePairTypeString = ResidueTypeString(p) + ResidueTypeString(q);\n\nResiduePairTypeStringSorted = Sort(ResiduePairTypeString;\n\n/ Reverse sign of potential difference accordingly /\n\nmultfactor = 1;\n\nif (ResiduePairTypeStringSorted != ResiduePairTypeString) then\n\nmultfactor = -1 ;\n\nend\n\n/ Let the amino acid pair be the kth in the set ϕpair /\n\nPD = PotentialDifference(p, q, atomP, atomQ) multfactor ;\n\nif (∃meanPDCk) then\n\nNumberCompared = NumberCompared + 1 ;\n\ndiff = absolute(PD − meanPDCk);\n\nPDscore = PDscore + diff;\n\nend\n\nend\n\n/* Normalize /\n\nPDscore = PDscore/NumberCompared;\n\nreturn (PDscore);\n\nend\n\nInput: ϕproteins: Learning set\n\nInput: P1: Protein to be scored\n\nInput: IgnoreNTerm: Ignore this number of residues in the N Terminal\n\nInput: IgnoreCTerm: Ignore this number of residues in the C Terminal\n\nInput: atomP: Atom type in first residue\n\nInput: atomQ: Atom type in second residue\n\nInput: sdThresh: Threshfold for standard deviation of the EPD\n\nOutput: PDscore: Score indicating the normalized distance of the observed values from the (mean)\n\nlearnt values from native structures\n\nbegin\n\n/ This is invoked once*/ ϕpairMean =\n\nLearnFeatures(ϕproteins, IgnoreNTerm, IgnoreCTerm, atomP, atomQ, sdT hresh);;\n\nPDscore = AssessEPDQuality(P1, IgnoreNTerm, IgnoreCTerm, atomP, atomQ, ϕpairMean);\n\nreturn (PDscore);\n\nend", "appendix": "Author contributions\n\n\n\nConceived and performed the experiments: SC. Analyzed the data, and improved experiments: SC BA AMD BJR RV. Wrote the manuscript: SC BA AMD BJR RV.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nBJ and RV acknowledge financial support from Tata Institute of Fundamental Research (Department of Atomic Energy). Additionally, BJR is thankful to the Department of Science and Technology for the JC Bose Award Grant. BA extends gratitude to the University of Iceland Research Found for supporting the project financially. AMD wishes to acknowledge grant #12-0130-SA from California Department of Food and Agriculture CDFA PD/GWSS Board.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe are grateful to Mary Lou Mendum critical reading of the manuscript.\n\n\nSupplementary Tables\n\nSet of 1000 proteins from the PISCES database with percentage identity cutoff of 20%, resolution cutoff of 1.6 Å, R-factor cutoff of 0.25, and a RDCC cutoff of 0.012 Å used to learn feature values.\n\nThe PDBs are sorted based on specificity: The ig_structal decoy set has ~61 protein structures - each of which has 61 structures. The average specificity obtained for the set is 0.97. NRes =: number of residues, NStructures =: number of structures in the decoy set.\n\n\nReferences\n\nZhang Y: Progress and challenges in protein structure prediction. Curr Opin Struct Biol. 2008; 18: 342–348. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "4062", "date": "12 Mar 2014", "name": "Shina Caroline Lynn Kamerlin", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting idea that uses the physical (electrostatic) properties of amino acid side chains in order to predict secondary structure from sequence, and thus assess (and rank) the quality of protein structures. The manuscript is well-written, and the authors provide comprehensive information to allow others to follow the study-design and methodology. The focus on electrostatics is important as this has been repeatedly shown by rigorous theoretical studies (work by Warshel and others) to be the primary driving force in determining protein function and most likely folding stability as well (whether directly or indirectly). As the specific methodology the authors use is slightly further from my area of expertise I cannot directly comment on this, however, importantly the source code has been made Open Access and freely available through Github.My only comment is on the second paragraph of the Discussion, which comments on the pitfalls when using an incorrect model of a protein structure, particularly when trying to calculate pKas using continuum models which are dependent on the initial conformation. While the authors highlight a very important challenge, I would like to point out that it can to some extent be resolved by extensive conformational sampling (particularly the pKa problem, as the pKa is an average property over all possible protein conformations), which we discussed at length in a review a few years ago (Kamerlin et al., 2009). Otherwise this is a nice manuscript and a valuable contribution to the literature.", "responses": [] }, { "id": "5376", "date": "18 Jul 2014", "name": "Patricia C Weber", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe quality of protein structure models is assessed by the geometry of adjacent C beta atoms. The approach successfully distinguishes properly folded proteins in most cases. It adds a new way to assess protein models and could be included in the protein model assessment toolbox.Just a few comments:a) Table 4 lists three of 20 structures where the incorrect one has a lower score. A few comments about structural features of those examples that lead unexpected scores would be useful.b) It might be preferable to note in the title that the method is applied to computational models of protein structure as a way to distinguish the manuscript from those that deal with quality assessment of experimentally determined structures.c) I did not test the available source code.", "responses": [ { "c_id": "985", "date": "15 Sep 2014", "name": "Sandeep Chakraborty", "role": "Author Response", "response": "We would like to thank you for taking the time and reviewing our paper, and deeply appreciate your positive comments. We do not have the expertise to comment on the structural characteristics that lead to the 3 negative results in Table 4 - these require insights into crystal structures that we do not possess at the present time. Also, we believe that this method could be applied to any structure - computational or experimentally obtained - and thus are leaving the title unchanged." } ] } ]	2	https://f1000research.com/articles/2-243
https://f1000research.com/articles/3-195/v1	14 Aug 14	{ "type": "Case Report", "title": "Case Report: Hyponatremia of malignancy – An alternative mechanism? Syndrome of inappropriate atrial natriuretic peptide (SIANP)", "authors": [ "Steven Elias Mansoor", "David I Kagen", "Devan Kansagara", "David I Kagen", "Devan Kansagara" ], "abstract": "Euvolemic hyponatremia in the setting of lung cancer is most commonly due to the syndrome of inappropriate anti-diuretic hormone secretion (SIADH). However, some patients with small cell carcinoma and hyponatremia have low levels of ADH but elevated levels of atrial natriuretic peptide (ANP), which is produced by some small cell tumors. We report the case of a 64-year-old man with a limited-stage small cell carcinoma of the lung undergoing chemoradiation therapy, who was admitted to hospital with a pulmonary embolism. Two months earlier, at the time of diagnosis with lung cancer, he had a hypotonic, euvolemic hyponatremia, presumed to be caused by SIADH. At that time, his serum sodium readily normalized with water restriction and ADH-antagonist therapy with demeclocycline. However, during his second admission, his sodium level slowly declined from 138 mmol/L to a nadir of 118 mmol/L, despite early initiation of water restriction and maximal doses of demeclocycline. Laboratory values revealed a very low level of ADH, an inappropriately low level of aldosterone and an elevated ANP suggesting that SIADH could not explain his hyponatremia. While a causal link between ectopic ANP production and hyponatremia has never been established, an inappropriately high level of ANP can directly decrease sodium re-absorption in the proximal convoluted tubule of the kidney and increase glomerular filtration rate (GFR), resulting in greater excretion of sodium and water. In addition, high circulating levels of ANP can inhibit aldosterone secretion, potentially resulting in further sodium wasting. Here, the low levels of ADH, elevated ANP, and inappropriately low aldosterone suggested the possibility of an ANP-mediated hyponatremia through the suppression of aldosterone response.", "keywords": [ "ADH", "SIADH", "ANP", "hyponatremia" ], "content": "Introduction\n\nHyponatremia is commonly found in patients that have been diagnosed with lung cancer – reportedly as high as 15–30% of patients with small cell lung carcinoma (SCLC) present with hyponatremia1,2. While it is clear that water and sodium homeostasis are abnormal in these patients, the complete pathogenesis controlling the hormonal mechanisms of renal sodium and water re-absorption in malignancy has not yet been completely elucidated. However, SCLC is associated with a variety of paraneoplastic syndromes characterized by the ectopic production of peptide hormones or centrally-active antibodies3,4. The most common of these paraneoplastic phenomena is hyponatremia of malignancy, traditionally thought to be caused by elevated levels of arginine vasopressin (AVP), otherwise known as anti-diuretic hormone (ADH), ectopically produced by the neoplastic cells. Inappropriately elevated ADH levels increase the permeability of water in the cells of the distal tubule and collecting duct of the kidney, increasing water re-absorption and decreasing free water clearance, thus resulting in subsequent hyponatremia. This condition is widely known as the syndrome of inappropriate ADH secretion or SIADH2,5,6, and antineoplastic therapy and methods to correct hyponatremia such as fluid and sodium restriction typically constitute effective treatment strategies.\n\nWhile SIADH is clearly responsible for the majority of cases of hyponatremia of malignancy, there have been documented cases of SCLC patients with hyponatremia, but with no detectable levels of ADH in their plasma7,8 or produced by their cancer cells9–11. These interesting observations led a number of researchers to investigate potential alternative mechanisms to explain the low levels of sodium observed in these cases. An alternative hypothesis based on reports showing ectopic production of atrial natriuretic peptide (ANP) mRNA in tumor lines, would suggest the possibility of an analogous ectopic hormone syndrome: elevated levels of ectopically produced ANP, referred to here as the syndrome of inappropriate ANP or SIANP. This hypothesis has been attractive because ANP is thought to have physiologic effects that promote natriuresis: direct sodium wasting effects on the kidney through ANP receptors, inhibition of renin secretion and inhibition of aldosterone secretion12–14. Despite the evidence for ectopic production of ANP in tumor cell lines, there is no evidence revealing a causal link between ANP and hyponatremia.\n\nHere we report the case of a middle-age man with known small cell carcinoma of the lung, who developed a progressive, profound hyponatremia despite strict fluid restriction and ADH-antagonist therapy. Laboratory analysis revealed elevated urine osmoles, very low levels of ADH, inappropriately low levels of aldosterone, and elevated serum ANP despite no known history of heart failure and no clinical signs of volume overload.\n\n\nCase description\n\nA 64-year-old man with limited-stage small cell carcinoma of the lung (Figure 1) undergoing concurrent chemoradiation therapy was admitted to hospital with a pulmonary embolism after collapsing en route to his seventh radiation treatment. At the time of admission, he was at 10 days status after his first cycle of carboplatin/etoposide chemotherapy and he had six treatments of radiation therapy. Admission laboratory values were notable only for a leukopenia and a serum sodium level of 138 mmol/L.\n\nThe numerous atypical small cells with minimal cytoplasm are characteristic of metastatic small cell carcinoma. Inset. Immunohistochemical staining is positive for the neuroendocrine marker, synaptophysin, further supporting the diagnosis.\n\nTwo months earlier, at the time of his small cell lung cancer diagnosis, he presented with an acute onset altered mental status. A work-up revealed a hypotonic, euvolemic hyponatremia (Na+ 116 mmol/L), with elevated urine osmolality (609 mOsm/Kg) and urine sodium (181 meq/L). He was presumptively diagnosed with SIADH syndrome, and was treated with a mild fluid restriction and ADH antagonist (demeclocycline at 300 mg PO BID) therapy (Figure 2A), which restored his normal serum sodium levels after < 3 weeks of therapy. Upon discharge from the hospital after his initial diagnosis, he was continued on 300 mg PO BID of demeclocycline, which he was taking at the time of the current admission for the pulmonary embolism. At home, he was not observing a fluid restricted diet.\n\n(A) Initial presentation and diagnosis of small cell carcinoma of the lung. (B) Two months later during the current hospitalization when he presented to our service. (A) At the time of initial hospitalization for altered mental status, the patient was found to have a sodium level of 116 and an elevated urine osmolality (609 mOsm/Kg). Water restriction and demeclocycline treatment resulted in a slow, steady rise in sodium back toward normal, consistent with SIADH. (B) On our service, a slow and steady decline in sodium level was observed despite the early initiation of demeclocycline treatment and sodium and fluid restriction (represented by red arrow). Starting on day 3, the patient resumed radiation therapy after going 5 days without receiving radiation. After reaching a minimum of 118 mmol/L, the sodium level began to rise toward normal. AVP, ADH, and aldosterone levels were drawn on day 13, represented by the blue arrow, and revealed an elevated AVP, but low ADH and low aldosterone (Table 1), consistent with hyponatremia due to SIANP but inconsistent with a hyponatremia due to SIADH.\n\nWhile in the hospital for treatment of the pulmonary embolism, his sodium level steadily declined from the admission value of 138 mmol/L to 118 mmol/L over 11 days (Figure 2B), despite severe fluid restriction and increases to maximal doses of demeclocycline (600 mg PO BID, initiated at the red arrow in Figure 2B). Of note, his physical exam remained consistent with a euvolemic hyponatremia – he did not have any evidence of jugular venous distention, pulmonary edema or lower extremity edema. His liver enzymes were within normal limits (AST = 22 IU/L, alkaline phosphatase = 88 IU/L, and total bilirubin = 0.4 mg/dL) and his kidney function remained normal (Cr = 0.7 mg/dL). Further, his thyroid stimulating hormone, free T4 and cortisol levels were normal (Table 1), and his urine osmolality (563 mOsm/Kg) and urine sodium (128 mmol/L) remained high. A computed tomography (CT) scan of the brain revealed no evidence of metastatic disease or intracranial hemorrhage.\n\nIn addition, the low ADH level rules out SIADH while the elevated ANP level and low aldosterone level are consistent with a hyponatremia due to SIANP.\n\nDuring the initial period of hospitalization, the patient was not able to undergo radiation treatments – this therapy resumed on hospital day 3 after having gone five full days without a treatment. Starting on hospital day 12, sodium levels began to spontaneously increase, returning to 135 mmol/L by day 15. On day 13, when the sodium levels were at 125 mmol/L, laboratory values were drawn in an attempt to qualify the etiology of the hyponatremia: ADH levels were 1.7 pg/mL (low < 7.0 pg/mL), aldosterone levels were < 3 ng/dL (low < 32 ng/dL) and ANP levels were at 140 pg/mL (normal 20 – 77 pg/mL) (see Table 1). The laboratory results, imaging data, and his physical exam findings suggested that neither SIADH nor any of the other common etiologies (hypovolemia, hypothyroidism, adrenal insufficiency, cerebral salt wasting) could explain this patient’s hyponatremia. Fortunately for the patient, once his sodium level began to trend upward, it stayed in the normal range for the duration of his hospital stay and was 137 mmol/L on discharge. Successful reversal of the hyponatremia was attributed to resumption of his radiation therapy with effective treatment of his malignancy.\n\n\nDiscussion\n\nIn 1957, Schwartz et al. presented the first cases of hyponatremia of malignancy from inappropriate anti-diuretic hormone secretion in two patients with lung cancer who developed low serum sodium levels associated with continued urinary sodium losses15. The authors correctly postulated that the tumors were producing excessive levels of anti-diuretic hormone (vasopressin) via a feedbackinsensitive mechanism, a hypothesis which was later proven16–18. The same group subsequently went on to further characterize the SIADH and proposed three mechanisms which could theoretically explain the ongoing urinary sodium losses5: (i) an increase in the glomerular filtration rate leading to an increase in the filtered load of sodium, (ii) a suppression of tubular re-absorption of sodium secondary to expansion of the extracellular fluid volume, and (iii) a suppression of aldosterone secretion as a result of the elevated extracellular fluid volume.\n\nIt is well now known that ADH acts primarily to insert aquaporin channels into the apical membrane of the distal tubule and collecting ducts of the kidney, increasing the permeability of these cells to water, and allowing its re-absorption and excretion of concentrated urine19–21. This process results in an ADH-induced water retention; the subsequent volume expansion activates the mechanisms for secondary natriuresis mentioned above, resulting in both a sodium and water loss in an attempt to restore euvolemia. With chronic SIADH, sodium loss is much more prominent than water retention and hyponatremia ensues. There has been much discussion on the diagnosis, pathophysiology, and management of hyponatremia secondary to SIADH2,6.\n\nHowever, it is interesting to note that as many as one-third of patients with documented small cell lung cancer and hyponatremia do not have elevated levels of serum ADH or ectopic production of ADH from their tumor cells1,7,22,23, leading some to propose the more appropriate umbrella term syndrome of inappropriate antidiuresis (SIAD), while reserving the term SIADH for only those cases where ADH levels are actually elevated6,24. An alternative etiology for hyponatremia of malignancy in patients without elevated ADH levels includes ectopic production of ANP, as we postulate in the case presented in this report. As mentioned above, there is evidence that ectopic production of ANP can contribute to hyponatremia.\n\nANP is a 28 amino acid peptide secreted from the atrial myocytes in response to local wall stretch and increased atrial volume, but also found in the hypothalamus, brainstem nuclei, pituitary and vascular tissue, kidney, and adrenal medulla12. It was identified and characterized after de Bold et al. demonstrated that a cardiac extract produced natriuresis in rats25. While the details surrounding all the physiologic roles of ANP are still under discussion, it seems that renal, vascular, and cardiac actions are important in maintaining the body fluid and sodium homeostasis12,13. The physiological effects of ANP are summarized in Figure 3. Overall, the main function of ANP is to counter increases in blood pressure and volume induced by the activation of the renin-angiotensin system. As such, the actions of ANP result in natriuresis.\n\nIn response to local wall stretch and increased atrial volume, atrial myocytes secrete ANP, which dilates smooth muscle and blocks the action of norepinephrine and angiotensin II, causing a prolonged increase in glomerular filtration rate (GFR). This causes afferent arteriolar dilation with or without efferent arteriolar constriction and the increased GFR increases the filtered load of sodium, washing out the sodium gradient. It has also been demonstrated that ANP directly increases renal excretion of sodium, acting through ANP receptors in the kidney at the distal convoluted tubule (DCT) and the collecting duct (CD). ANP also directly decreases production of renin, angiotensin II and aldosterone contributing to hyponatremia through natriuresis, negative sodium balance, and non-osmotic release of arginine vasopressin (AVP) (from decreased intravascular volume).\n\nThis is accomplished through three distinct but inter-related mechanisms. First, ANP is known to dilate smooth muscle in arterioles and venules and block the action of norepinephrine, causing vasodilatation and increased renal blood flow12,13. This effectively increases the glomerular filtration rate (GFR) in the kidney leading to increases in the filtered load of sodium and subsequent natriuresis13. Radionucleotide studies performed in humans support these results by showing both a significant decrease in the mean blood pressure as well as an increase in GFR after intravenous infusion of ANP peptide26. Second, ANP acts directly on ANP receptors in the kidney to increase renal excretion of sodium9,12. This is thought to occur via receptor-induced increases in cyclic GMP which directly increases GFR, inhibits secretion of renin, and reduces sodium and water re-absorption in the collecting duct9,13. These data have been supported by experiments in rats that demonstrated that urine flow and sodium excretion decreases while plasma renin increases after administration of antibodies raised against ANP27. Finally, it is thought that ANP directly blocks the production of renin, angiotensin II, and aldosterone, as these hormones showed decreased levels following ANP intravenous infusions12,14,28. Suppression of aldosterone secretion from the adrenal medulla is yet another mechanism by which ANP can contribute to natriuresis and subsequent hyponatremia.\n\nThese physiologic effects suggest that ANP could feasibly be implicated in cases of hyponatremia of malignancy without ectopic production of ADH. Further support for this hypothesis arose when researchers demonstrated ectopic production of ANP mRNA in both tumors and tumor cells of patients with hyponatremia of malignancy from small cell carcinoma. This has been shown using a number of techniques including northern blot, PCR, and nuclease protection assays1,7,23, and the ANP peptide itself has been detected in small cell lung cancer tumors7,22,29,30. Further, there are reports of the resolution of clinical hyponatremia following surgical resection of an ANP expressing tumor22. Interestingly, plasma levels of ANP have also been found to be elevated in patients with lung carcinoma and elevated plasma levels of ADH1,22,30. A recent case report presented a patient with SCLC, elevated levels of ANP, and elevated levels of ADH that varied in an oscillatory manner31.\n\nDespite the reports demonstrating the association of ectopic ANP production and hyponatremia, there is still not overwhelming evidence proving a causal relationship. An early prospective study trying to establish whether ectopically produced ANP could contribute to hyponatremia through natriuresis failed to show a correlation between plasma ANP levels and levels of plasma renin, angiotensin II, and aldosterone. The authors concluded that ANP was unlikely to contribute to hyponatremia via suppression of the renin-angiotensin II-aldosterone axis1. In this study, there were 14 patients with elevated levels of ANP but only three had small cell carcinoma associated with hyponatremia. It is not clear why no correlation was found, however, the same group later published a study where four patients with SCLC and elevated ANP levels had inappropriately low levels of aldosterone9. In this second study, the patients had persistent natriuresis and low serum aldosterone despite decreasing serum sodium levels while being treated with a fluid- and sodium-restricted diet. Thus, the normal physiological aldosterone response failed to occur in patients with elevated ANP9. From this study, the authors were able to make three major conclusions: (i) hyponatremia in SCLC patients is often associated with inappropriate elevations of ANP instead of elevations in AVP; (ii) SCLC patients with elevated level of ANP do not seem to respond to fluid restriction, and in fact, fluid restriction may worsen the hyponatremia; and (iii) ANP appears to mediate some of its natriuretic effects through suppression of aldosterone. Interestingly, the patient presented in this manuscript displayed a disease process which exactly fits this description: elevated levels of ANP without elevated levels of ADH, a worsening hyponatremia despite strict fluid and sodium restriction, and inappropriately low levels of aldosterone.\n\nHyponatremia potentially caused by SIANP should not be expected to respond to the traditional treatments used for SIADH. As discussed above, water restriction, beneficial in SIADH, may worsen the hyponatremia of SIANP if sodium intake is not increased concurrently. Likewise, if ADH levels are already suppressed in SIANP, ADH antagonists such as demeclocycline or conivaptan will have no benefits. To date, no ANP antagonists have been developed and there is no specific treatment for SIANP except for addressing the underlying cause. However, it is still important to distinguish between SIANP and SIADH. Certainly, any patient presenting with hyponatremia from SCLC should initially be placed on fluid and sodium restriction because SIADH seems to be more common. However, if the hyponatremia continues to worsen after 48–96 hours, alternative etiologies including ectopic ANP production should be considered and plasma levels of ANP and AVP should be measured to confirm the underlying etiology of the hyponatremia. It is very important to note that in the current case, the spontaneous correction of hyponatremia starting on hospital day 12 was temporally associated with appropriate tumor response to radiation therapy and, perhaps, an associated reduction in circulating levels of ectopic ANP. This suggests that for the time being, in the cases of SIANP, early treatment of the underlying malignancy might be the best way to correct the underlying hyponatremia.", "appendix": "Author contributions\n\n\n\nSEM admitted the patient, challenged the original diagnosis of SIADH, and made the diagnosis of SIANP. SEM made all the figures and wrote the manuscript. DIK and DK were the Attending Physicians on service for the care of this patient. DIK and DK helped design Figure 3. All authors read and approved the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe thank Dr. Michael Bliziotes, Dr. Lynn Loriaux, and Dr. David B. Jacoby for the critical reading of this manuscript.\n\n\nReferences\n\nJohnson BE, Chute JP, Rushin J, et al.: A prospective study of patients with lung cancer and hyponatremia of malignancy. Am J Respir Crit Care Med. 1997; 156(5): 1669–1678. PubMed Abstract \| Publisher Full Text\n\nSorensen JB, Andersen MK, Hansen HH: Syndrome of inappropriate secretion of antidiuretic hormone (SIADH) in malignant disease. J Intern Med. 1995; 238(2): 97–110. PubMed Abstract \| Publisher Full Text\n\nKazarian M, Laird-Offringa IA: Small-cell lung cancer-associated autoantibodies: potential applications to cancer diagnosis, early detection, and therapy. Mol Cancer. 2011; 10: 33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcClelland MT: Paraneoplastic syndromes related to lung cancer. Clin J Oncol Nurs. 2010; 14(3): 357–364. PubMed Abstract \| Publisher Full Text\n\nBartter FC, Schwartz WB: The syndrome of inappropriate secretion of antidiuretic hormone. Am J Med. 1967; 42(5): 790–806. PubMed Abstract \| Publisher Full Text\n\nEllison DH, Berl T: Clinical practice. The syndrome of inappropriate antidiuresis. N Engl J Med. 2007; 356(20): 2064–2072. PubMed Abstract \| Publisher Full Text\n\nBliss DP Jr, Battey JF, Linnoila RI, et al.: Expression of the atrial natriuretic factor gene in small cell lung cancer tumors and tumor cell lines. J Natl Cancer Inst. 1990; 82(4): 305–310. PubMed Abstract \| Publisher Full Text\n\nBondy PK, Gilby ED: Endocrine function in small cell undifferentiated carcinoma of the lung. Cancer. 1982; 50(10): 2147–2153. 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PubMed Abstract\n\nMaack T: Role of atrial natriuretic factor in volume control. Kidney Int. 1996; 49(6): 1732–1737. PubMed Abstract \| Publisher Full Text\n\nSchwartz WB, Bennett W, Curelop S, et al.: A syndrome of renal sodium loss and hyponatremia probably resulting from inappropriate secretion of antidiuretic hormone. Am J Med. 1957; 23(4): 529–542. PubMed Abstract \| Publisher Full Text\n\nAmatruda TT Jr, Mulrow PJ, Gallagher JC, et al.: Carcinoma of the Lung with Inappropriate Antidiuresis. Demonstration of Antidiuretic-Hormone-Like Activity in Tumor Extract. N Engl J Med. 1963; 269: 544–549. PubMed Abstract \| Publisher Full Text\n\nGeorge JM, Capen CC, Phillips AS: Biosynthesis of vasopressin In vitro and ultrastructure of a bronchogenic carcinoma. Patient with the syndrome of inappropriate secretion of antidiuretic hormone. J Clin Invest. 1972; 51(1): 141–148. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKlein LA, Rabson AS, Worksman J: In vitro synthesis of vasopressin by lung tumor cells. Surg Forum. 1969; 20: 231–233. PubMed Abstract\n\nTakata K, Matsuzaki T, Tajika Y, et al.: Localization and trafficking of aquaporin 2 in the kidney. Histochem Cell Biol. 2008; 130(2): 197–209. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIshikawa SE, Schrier RW: Pathophysiological roles of arginine vasopressin and aquaporin-2 in impaired water excretion. Clin Endocrinol (Oxf). 2003; 58(1): 1–17. PubMed Abstract \| Publisher Full Text\n\nIshikawa S: Cellular actions of arginine vasopressin in the kidney. Endocr J. 1993; 40(4): 373–386. PubMed Abstract \| Publisher Full Text\n\nCampling BG, Sarda IR, Baer KA, et al.: Secretion of atrial natriuretic peptide and vasopressin by small cell lung cancer. Cancer. 1995; 75(10): 2442–2451. PubMed Abstract \| Publisher Full Text\n\nGross AJ, Steinberg SM, Reilly JG, et al.: Atrial natriuretic factor and arginine vasopressin production in tumor cell lines from patients with lung cancer and their relationship to serum sodium. Cancer Res. 1993; 53(1): 67–74. PubMed Abstract\n\nFeldman BJ, Rosenthal SM, Vargas GA, et al.: Nephrogenic syndrome of inappropriate antidiuresis. N Engl J Med. 2005; 352(18): 1884–1890. PubMed Abstract \| Publisher Full Text\n\nde Bold AJ, Borenstein HB, Veress AT, et al.: A rapid and potent natriuretic response to intravenous injection of atrial myocardial extract in rats. Life Sci. 1981; 28(1): 89–94. PubMed Abstract \| Publisher Full Text\n\nCuocolo A, Volpe M, Mele AF, et al.: Effects of atrial natriuretic peptide on glomerular filtration rate in essential hypertension: a radionuclide study. Eur J Nucl Med. 1991; 18(1): 32–37. PubMed Abstract \| Publisher Full Text\n\nNaruse M, Obana K, Naruse K, et al.: Antisera to atrial natriuretic factor reduces urinary sodium excretion and increases plasma renin activity in rats. Biochem Biophys Res Commun. 1985; 132(3): 954–960. PubMed Abstract \| Publisher Full Text\n\nGaillard CA, Koomans HA, Rabelink AJ, et al.: Renal response to infusion versus repeated bolus injections of atrial natriuretic factor in man. Eur J Clin Pharmacol. 1989; 36(2): 195–197. PubMed Abstract \| Publisher Full Text\n\nJohnson BE, Damodaran A, Rushin J, et al.: Ectopic production and processing of atrial natriuretic peptide in a small cell lung carcinoma cell line and tumor from a patient with hyponatremia. Cancer. 1997; 79(1): 35–44. PubMed Abstract \| Publisher Full Text\n\nShimizu K, Nakano S, Nakano Y, et al.: Ectopic atrial natriuretic peptide production in small cell lung cancer with the syndrome of inappropriate antidiuretic hormone secretion. Cancer. 1991; 68(10): 2284–2288. PubMed Abstract \| Publisher Full Text\n\nRadulescu D, Bunea D, Pripon S, et al.: Severe paraneoplastic hyponatremia and hypoosmolality in a patient with small-cell lung carcinoma: syndrome of inappropriate antidiuretic hormone secretion versus atrial natriuretic peptide or both? Clin Lung Cancer. 2007; 8(6): 392–395. PubMed Abstract \| Publisher Full Text" }	[ { "id": "5837", "date": "15 Aug 2014", "name": "Richard Sterns", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe Case Report is extremely well written but, unfortunately, I feel that the basic premise of the paper is fundamentally flawed. The authors argue that their patient with SCLC and hyponatremia did not have SIADH because plasma ADH levels were not elevated. Since ANP levels were high, the authors propose that ectopic secretion of ANP was the cause of hyponatremia. There are several problems with their argument. First, unless done in a research laboratory, plasma ADH levels are extremely unreliable and are not recommended. Second, even if they were measured correctly, ADH levels do not have to be “high” to be abnormal in patients with hyponatremia; they merely have to be measurable , because ADH should be unmeasurable if the serum sodium is less than 135. Third, even if ectopic ANP were the primary cause of hyponatremia, ADH levels would still be expected to be elevated secondarily because hypovolemia resulting from ANP-induced sodium wasting should stimulate ADH secretion from the posterior pituitary; i.e. ADH is inappropriately high, but not ectopically secreted. Finally, like ADH levels, the finding of elevated ANP levels does not prove ectopic secretion (though the authors are correct that ectopic secretion of ANP is common in SCLC). ANP is secondarily high in all causes of SIADH, because water retention expands plasma volume which results in ANP secretion and secondary natriuresis. The fact that their patient was clinically euvolemic is more consistent with secondary secretion of ANP in response to water retention from SIADH. If the primary problem were ectopic ANP and inappropriate natriuresis, the patient should have looked hypovolemic. The fact that their patient failed to respond to demeclocycline is not definitive – some patients may not respond to maximum doses; furthermore, response to the drug would not distinguish between SIADH and secondary secretion of ADH due to ANP-induced hypovolemia. The paper would have been more interesting if the authors had arranged for measurement of vasopressin levels in a research laboratory and if they had treated their patient with V2-receptor antagonist. There are reported cases of hyponatremia with low levels of ADH, raising the possibility that some tumors secrete a substance that is not detected immunologically as ADH, but which acts as an antidiuretic hormone. The high Uosm is evidence that their patient was secreting an antidiuretic hormone – they might not have been able to measure it because of problems with their assay or because their patient was secreting an antidiuretic hormone immunologically distinct from vasopressin; unfortunately we can’t tell which is true.\n\nIn addition to the fundamental flaw that I see in the authors’ argument, they make some statements that I don’t agree with. For example, they say that water restriction would worsen hyponatremia caused by primary sodium loss. Regardless of the underlying causes, the serum sodium falls because net water intake exceeds net water loss. All else being equal, restriction of water intake always makes the serum sodium go up. If the patient is hypovolemic, the serum sodium concentration will rise with water restriction but hypovolemia will worsen.", "responses": [] }, { "id": "5924", "date": "26 Aug 2014", "name": "Michael Emmett", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper reports a patient with small cell cancer and recurrent episodes of hyponatremia. The first episode was entirely consistent with SIADH and responded to water restriction and demeclocycline. The second episode of hyponatremia occurred with a nadir Na of 118 and an “inappropriately” concentrated urine of 563 and a high urine Na of 128 mm/l. The measured ADH level was low and his aldosterone level was also low. The patient seemed to be euvolemic on exam. Clearly this patient had inappropriate urine concentration with a high urine osmolality, clinical euvolemia, and a urine Na of 128. The question is why. The single serum ADH level was low but is it correct? If it is truly low (and a level of 1.7 may still be too high for a plasma Na of 125) then something else is driving urine concentration. My understanding of ANP is in agreement with the author’s figure 3: it increases renal blood flow, GFR, and causes natiuresis. But it does not directly generate a concentrated urine. If ANP causes too much natiuresis (ie an “inappropriate ANP” syndrome) then the urine will become concentrated as a result of ECF volume contraction and high ADH levels. If the authors are proposing that “inappropriate ANP” syndrome can generate hyponatremia independent of elevated ADH levels this would be a new physiologic principal and I need a lot more evidence that this can occur than what is presented in this manuscript.", "responses": [] } ]	1	https://f1000research.com/articles/3-195
https://f1000research.com/articles/2-261/v1	28 Nov 13	{ "type": "Research Article", "title": "Transitory expression of Dlx5 and Dlx6 in maxillary arch precursors is essential for upper jaw morphogenesis", "authors": [ "Yorick Gitton", "Nicolas Narboux-Nême", "Giovanni Levi", "Yorick Gitton", "Nicolas Narboux-Nême" ], "abstract": "Asymmetric, articulated jaws support active predation in vertebrates; they derive from the first pharyngeal arch (PA1) which generates both maxillary and mandibular components. PA1 is colonized by cranial neural crest cells (CNCCs) which give rise to most bones and tendons of the jaws. The elements formed by different CNCCs contingents are specified by the combinatorial expression of Dlx genes. Dlx5 and Dlx6 are predominantly expressed by mandibular CNCCs. Analysis of the phenotype of Dlx5 and Dlx6 double mutant mice has suggested that they are necessary and sufficient to specify mandibular identity. Here, using 3D reconstruction, we show that inactivation of Dlx5 and Dlx6 does not only affect the mandibular arch, but results in the simultaneous transformation of mandibular and maxillary skeletal elements which assume a similar morphology with gain of symmetry. As Dlx5- and Dlx6-expressing cells are not found in the maxillary bud, we have examined the lineage of Dlx5-expressing progenitors using an in vivo genetic approach. We find that a contingent of cells deriving from precursors transiently expressing Dlx5 participate in the formation of the maxillary arch. These cells are mostly located in the distal part of the maxillary arch and might derive from its lambdoidal junction with the olfactory pit. Our findings extend current models of jaw morphogenesis and provide an explanation for the maxillary defects of Dlx5 and Dlx6 mutants. Our results imply that Dlx5 and Dlx6 model the upper and the lower PA1 components through different morphogenetic mechanisms which are, however, coordinated as they give rise to functional, articulated jaws.", "keywords": [ "The vertebrate skull is characterized by the presence of articulated", "asymmetric jaws which support the function of a muscularized oral cavity essential for predation. During embryonic development", "the upper and lower jaws derive from the maxillary and mandibular processes of the first pharyngeal arch (PA1). Most cartilaginous and dermatocranial derivatives of PA1 are formed by Cranial Neural Crest Cells (CNCCs) emigrating from the prosencephalic and anterior mesencephalic neural folds1–6. During migration", "signals emanating from the endoderm and possibly other PA1 components instruct the CNCCs to unfold the morphogenetic process of the jaws5", "7", "8. The nested expression of Dlx homeobox genes", "vertebrate homologues of Drosophila Distal-less", "has a fundamental role in the specification of the dorsoventral patterning of PA1 derivatives9", "10. The six Dlx genes found in mammals are arranged as closely associated bigenic clusters: Dlx1", "Dlx2", "Dlx3", "Dlx4", "Dlx5", "Dlx6", "these pairs of genes are often coregulated and display a partially redundant function. While Dlx1 and Dlx2 are expressed by CNCCs of the maxillary and mandibular components of PA1", "Dlx5 and Dlx6 transcripts are present only in mandibular CNCCs. Targeted simultaneous inactivation of Dlx5 and Dlx6 results in the transformation of lower jaw into upper jaw-like structures", "underlining the importance of these genes for lower jaw identity11–14. Interestingly it has been observed15", "16 that", "after inactivation of Dlx5 and Dlx6", "the maxillary component is also affected despite the fact that these genes are not expressed in maxillary CNCCs. This observation could be accounted for by the presence of shared Dlx5/6-dependent signalling centres in proximity to the extremities of both the mandibular and maxillary arches", "this notion gave rise to the so-called “hinge and caps” model of jaw organization17. In its original formulation this model predicts the presence of two opposing morphogen gradients", "one emanating from the region of the upper/lower jaw articulation (hinge) and one from the distal extremities of PA1 (caps). While the origin and nature of these signals remain elusive", "the possibility that transient Dlx expression in a contingent of cells populating the maxillary arch could play a role in its morphogenesis has not been yet analyzed. Here we revisit the effects of Dlx5 and Dlx6 double inactivation on jaw development and", "using a transgenic lineage tracing approach", "we reveal that the maxillary arch harbours a cellular contingent derived from Dlx5 progenitors." ], "content": "Introduction\n\nThe vertebrate skull is characterized by the presence of articulated, asymmetric jaws which support the function of a muscularized oral cavity essential for predation. During embryonic development, the upper and lower jaws derive from the maxillary and mandibular processes of the first pharyngeal arch (PA1). Most cartilaginous and dermatocranial derivatives of PA1 are formed by Cranial Neural Crest Cells (CNCCs) emigrating from the prosencephalic and anterior mesencephalic neural folds1–6. During migration, signals emanating from the endoderm and possibly other PA1 components instruct the CNCCs to unfold the morphogenetic process of the jaws5,7,8. The nested expression of Dlx homeobox genes, vertebrate homologues of Drosophila Distal-less, has a fundamental role in the specification of the dorsoventral patterning of PA1 derivatives9,10. The six Dlx genes found in mammals are arranged as closely associated bigenic clusters: Dlx1, Dlx2; Dlx3, Dlx4; Dlx5, Dlx6; these pairs of genes are often coregulated and display a partially redundant function. While Dlx1 and Dlx2 are expressed by CNCCs of the maxillary and mandibular components of PA1, Dlx5 and Dlx6 transcripts are present only in mandibular CNCCs. Targeted simultaneous inactivation of Dlx5 and Dlx6 results in the transformation of lower jaw into upper jaw-like structures, underlining the importance of these genes for lower jaw identity11–14. Interestingly it has been observed15,16 that, after inactivation of Dlx5 and Dlx6, the maxillary component is also affected despite the fact that these genes are not expressed in maxillary CNCCs. This observation could be accounted for by the presence of shared Dlx5/6-dependent signalling centres in proximity to the extremities of both the mandibular and maxillary arches; this notion gave rise to the so-called “hinge and caps” model of jaw organization17. In its original formulation this model predicts the presence of two opposing morphogen gradients, one emanating from the region of the upper/lower jaw articulation (hinge) and one from the distal extremities of PA1 (caps). While the origin and nature of these signals remain elusive, the possibility that transient Dlx expression in a contingent of cells populating the maxillary arch could play a role in its morphogenesis has not been yet analyzed. Here we revisit the effects of Dlx5 and Dlx6 double inactivation on jaw development and, using a transgenic lineage tracing approach, we reveal that the maxillary arch harbours a cellular contingent derived from Dlx5 progenitors.\n\n\nMaterial and methods\n\nAll animal experimentation was performed in accordance to French national regulations and approved by the MNHN ethical committee (approval n° 68–028r1). For this study we used about 35 dams (including 10 WT, 5 Dlx5lacZ/+; 3 Dlx5/6+/-; 12 B6.129S4-Gt(ROSA)26Sortm1Sor/J; 5 B6(Cg)-Dlx5tm1(cre/ERT2)Zjh/J) and analyzed about 120 embryos, the exact record of animals used, litters obtained, embryos genotyped and number of embryos per litter is on record in our animal house. WT animals were form Charles River France and were maintained in the MNHN mouse facility which is officially certified by the French National Animal well being committee.\n\nDlx5lacZ/+ knock-in mice were maintained on a mixed B6/D2 genetic background18. Double Dlx5 and Dlx6 (Dlx5/6) mutant mice were maintained and genotyped as reported19. The inducible Cre driver strain B6(Cg)-Dlx5tm1(cre/ERT2)Zjh/J (designed by Z. J. Huang20), and the lacZ Cre reporter strain B6.129S4-Gt(ROSA)26Sortm1Sor/J (R26R-lacZ) were purchased from Jackson Laboratory (#10705 and #003309 respectively; Maine, USA) through Charles River Laboratories (L’Arbresle, France) and maintained on a C57BL/6J genetic background through heterozygous mating. Double heterozygous embryos were obtained through bi-directional crosses. Induction of Cre recombinase activity was obtained upon single intraperitoneal injection of 5mg of tamoxifen (Sigma-Aldrich), in corn oil. Tamoxifen preparation and administration in pregnant dams followed the Jackson Laboratory’s Guidelines and CNRS/MNHN Animal Handling Guidelines. Dams were euthanized by cervical dislocation at indicated stages and embryos were collected in phosphate-buffered saline (PBS), then staged and fixed by immersion in ice-cold fixative (2% paraformaldehyde/0.2% glutaraldehyde) for 5 to 15 minutes (depending upon their developmental stage).\n\nFor lacZ expression, embryos were fixed for 15–30 min in 4% paraformaldehyde; X-gal staining was performed as described previously18,21. Vehicle (corn oil) injection in double heterozygous mice did not yield leaking ß-galactosidase activity.\n\nHeads from 18.5 dpc (days post coitum) Dlx5/6-/- and wild type mouse embryos were fixed in Bouin’s solution (Sigma, France), embedded in paraffin and complete sets of frontal or parasagittal serial sections (12µm) were prepared. All sections were stained by Mallory’s trichrome as in22 and photographed (Nikon Digital Site DS-FI1). Pictures were aligned, piled and registered using the Fiji plug-in of NIH ImageJ \"Register Virtual Stack Slices\" (http://fiji.sc/wiki/index.php/Register_Virtual_Stack_Slices). 3D segmentation was performed with Mimics (Materialise, Belgium: http://biomedical.materialise.com/mimics) and visualized using Adobe Acrobat 9 pro.\n\n\nResults\n\nPrevious reports suggest that double inactivation of Dlx5 and Dlx6 results in lower-to-upper jaw transformation; these reports also indicated that the upper jaw of these mice is not normal15,16. To better visualize the jaw phenotype of Dlx5/6 mutants, we performed 3D reconstruction of craniofacial elements of 18.5 dpc (days post coitum) embryos. Frontal view of the mutant jaws (Figure 1, upper panel) shows an obvious gain of symmetry compared to a WT animal. Examining the defects of the lower and upper jaws separately (Figure 1, middle and lower panels), it is evident that both are transformed. In the absence of Dlx5 and Dlx6 the dentary and the upper jaw bones do not form correctly and are replaced by remarkably similar skeletal structures. In the mutant embryos, both the upper and lower jaw skeletal elements are reduced in size, are not fused in the midline, and display a lateral process positionally homologous to the wild type zygomatic arch. Thus the upper and lower jaw mutant bones resemble each other more closely than usually found in their normal counterparts.\n\nUpper row: Frontal view of WT and Dlx5/6-/- oral apparatus. Skeletal elements are grey, the tongue is red and incisors are purple. Middle row: Dorsal view of the dentary bone of WT and Dlx5/6-/- 18.5 dpc mice. Lower row: Ventral view of the maxillary components of WT and Dlx5/6-/- 18.5 dpc mice. Note that the inactivation of Dlx5/6 results in the transformation of both lower and upper jaw skeletal elements into new structures which appear more similar to each other than to their WT counterpart. cp, coronoid processes; dt, dentary bone; li, lower incisor; t, tongue; ui, upper incisor; za, zygomatic arch; za*, zygomatic arch-like structure deriving from lower jaw transformation; za’, zygomatic arch-like structure deriving from upper jaw transformation.\n\nIn Dlx5-lacZ heterozygous Theiler stage (ts) 19 (12 dpc) embryos the reporter is active in the olfactory pit and mandibular arch, but not in the maxillary arch; this pattern of expression does not change upon tamoxifen treatment of the pregnant dam (Figure 2A,A’). To understand the origin of the Dlx5/6-dependent defect of the upper jaw we used a genetic approach to follow the lineage of Dlx5-precursors in the head. To this end we brought the R26R-lacZ reporter into the Dlx5-creERT2 driver background and we activated cre-recombinase activity by tamoxifen treatment of the pregnant dam at ts9 (6.25 dpc). We monitored ß-gal reporter activity from ts15 (10 dpc) to ts20 (12.5 dpc). At ts15 we observed a stream of ß-gal-positive cells extending from the lambdoidal junction, which joins the olfactory pit with the distal maxillary arch17,23, towards the body of the maxillary arch (Figure 2B,B’). At ts19 and ts20 (Figure 2C,C’; D,D’) reporter-expressing cells are found in the upper epithelial lining of the maxillary arch (arrowheads in Figure 2C’,2D’) and in two distinct proximal and distal territories of the arch body (red asterisk in Figure 2C’).\n\nß-Galactosidase activity in the cephalic region of Dlx5-lacZ (A,A’) and Dlx5-creERT2; R26R-lacZ mouse embryos (B–D’). In all cases pregnant dams were treated with tamoxifen at 6.25 dpc/Theiler stage 9 (ts9) and embryos were collected at the indicated Theiler stage. (A,A’) As expected, even after tamoxifen treatment, Dlx5 is expressed in the mandibular arch (md), in the olfactory pit (olf), in the otic vesicle (ov), in the basal telencephalon (bt) and in the hind limb (hl), but not in the maxillary arch. (B,B’) Permanent activation of lacZ reporter expression in derivatives of Dlx5-expressing early progenitors (ts9) reveals the presence of a positive cellular contingent in the ts15 lambdoidal junction (λ) between the olfactory pit and the maxillary process. (C,C’; D,D’) At later developmental stages (ts19, ts20) a contingent of lacZ positive cells populates the distal domain of the maxillary arch. hl, hind limb; md, mandibular arch; mx, maxillary arch; olf, olfactory pit; ov, otic vesicle; bt, basal telencephalon; λ, lambdoidal junction; red asterisk/black arrowheads, territories of the maxillary arch colonized by derivatives of Dlx5-expressing progenitors. Bar: A–D 1mm; A’–D’ 250µm.\n\n\nDiscussion\n\nIn this study we have re-examined the skeletal jaw phenotype of Dlx5/6 mutant mice. We confirm that both the mandibular and maxillary arches are transformed. The profound change in the shape of the maxillary arch is difficult to explain as this region does not derive from a Dlx5/6-expressing territory. Lineage analysis to identify derivatives of Dlx5-positive progenitors reveals a new population of cells extending from the olfactory pit through the lambdoidal junction towards the maxillary arch17,23. These derivatives of Dlx5-positive cells have lost Dlx5 expression as seen by Dlx5 in situ hybridization (see for example Depew et al. (2002)16, Acampora et al. (1999)18 and Depew et al. (1999)24) and by lacZ-Dlx5 knock-in18, and Figure 2A’. We have shown that early Dlx5 and Dlx6 expression in the anterior neural fold is essential for nasal capsule patterning25; our present findings suggest that the same population of cells could also contribute to maxillary patterning. This cell contingent might well exert a patterning role upon the maxillary arch providing either epithelial or mesenchymal cues. This observation fits with the prediction of the ‘hinge and caps’ model17, and suggests that ’cap’ signals could originate from derivatives of Dlx5-expressing progenitors. Even if after migration in the maxillary arch these cells lose Dlx5 expression, it is still possible that the early expression of Dlx5 confers on them the capacity to pattern maxillary arch CNCCs, which do not themselves express Dlx5 and Dlx6. In contrast, in the lower jaw Dlx5 and Dlx6 are expressed by CNCCs; it appears, therefore, that Dlx5 and Dlx6 pattern the upper and lower jaw through very different mechanisms, which must be coordinated to generate the asymmetric, articulated, muscularized jaws of vertebrate predators.", "appendix": "Author contributions\n\n\n\nGL and YG conceived the study and designed the experiments. YG and NN-N carried out the research. GL and YG prepared the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was partially supported by the EU Consortium IDEAL (HEALTH-F2-2011-259679) to GL.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThis work was made possible thanks to the excellent technical assistance of Mss. Anastasia Fontaine, Aurélie Hagneau, Ocilia Fernandes and Gladys Alfama.\n\n\nReferences\n\nTan SS, Morriss-Kay GM: Analysis of cranial neural crest cell migration and early fates in postimplantation rat chimaeras. J Embryol Exp Morphol. 1986; 98: 21–58. PubMed Abstract\n\nCouly GF, Coltey PM, Le Douarin NM: The triple origin of skull in higher vertebrates: a study in quail-chick chimeras. Development. 1993; 117(2): 409–29. PubMed Abstract\n\nCreuzet S, Couly G, Vincent C, et al.: Negative effect of Hox gene expression on the development of the neural crest-derived facial skeleton. Development. 2002; 129(18): 4301–13. PubMed Abstract\n\nKontges G, Lumsden A: Rhombencephalic neural crest segmentation is preserved throughout craniofacial ontogeny. Development. 1996; 122(10): 3229–42. PubMed Abstract\n\nNoden DM: Vertebrate craniofacial development: novel approaches and new dilemmas. Curr Opin Genet Dev. 1992; 2(4): 576–81. PubMed Abstract \| Publisher Full Text\n\nTrainor PA, Tam PP: Cranial paraxial mesoderm and neural crest cells of the mouse embryo: co-distribution in the craniofacial mesenchyme but distinct segregation in branchial arches. Development. 1995; 121(8): 2569–82. PubMed Abstract\n\nRuhin B, Creuzet S, Vincent C, et al.: Patterning of the hyoid cartilage depends upon signals arising from the ventral foregut endoderm. Dev Dyn. 2003; 228(2): 239–46. PubMed Abstract \| Publisher Full Text\n\nCouly G, Creuzet S, Bennaceur S, et al.: Interactions between Hox-negative cephalic neural crest cells and the foregut endoderm in patterning the facial skeleton in the vertebrate head. Development. 2002; 129(4): 1061–73. PubMed Abstract\n\nMerlo GR, Zerega B, Paleari L, et al.: Multiple functions of Dlx genes. Int J Dev Biol. 2000; 44(6): 619–26. PubMed Abstract\n\nDepew MJ, Simpson CA, Morasso M, et al.: Reassessing the Dlx code: the genetic regulation of branchial arch skeletal pattern and development. J Anat. 2005; 207(5): 501–61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClouthier DE, Hosoda K, Richardson JA, et al.: Cranial and cardiac neural crest defects in endothelin-A receptor-deficient mice. Development. 1998; 125(5): 813–24. PubMed Abstract\n\nOzeki H, Kurihara Y, Tonami K, et al.: Endothelin-1 regulates the dorsoventral branchial arch patterning in mice. Mech Dev. 2004; 121(4): 387–95. PubMed Abstract \| Publisher Full Text\n\nRuest LB, Xiang X, Lim KC, et al.: Endothelin-A receptor-dependent and -independent signaling pathways in establishing mandibular identity. Development. 2004; 131(18): 4413–23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFukuhara S, Kurihara Y, Arima Y, et al.: Temporal requirement of signaling cascade involving endothelin-1/endothelin receptor type A in branchial arch development. Mech Dev. 2004; 121(10): 1223–33. PubMed Abstract \| Publisher Full Text\n\nBeverdam A, Merlo GR, Paleari L, et al.: Jaw transformation with gain of symmetry after Dlx5/Dlx6 inactivation: mirror of the past? Genesis. 2002; 34(4): 221–7. PubMed Abstract \| Publisher Full Text\n\nDepew MJ, Lufkin T, Rubenstein JL: Specification of jaw subdivisions by Dlx genes. Science. 2002; 298(5592): 381–5. PubMed Abstract \| Publisher Full Text\n\nDepew MJ, Compagnucci C: Tweaking the hinge and caps: testing a model of the organization of jaws. J Exp Zoolog B Mol Dev Evol. 2008; 310(4): 315–35. PubMed Abstract \| Publisher Full Text\n\nAcampora D, Merlo GR, Paleari L, et al.: Craniofacial, vestibular and bone defects in mice lacking the Distal-less-related gene Dlx5. Development. 1999; 126(17): 3795–809. PubMed Abstract\n\nMerlo GR, Paleari L, Mantero S, et al.: Mouse model of split hand/foot malformation type I. Genesis. 2002; 33(2): 97–101. PubMed Abstract \| Publisher Full Text\n\nTaniguchi H, He M, Wu P, et al.: A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex. Neuron. 2011; 71(6): 995–1013. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGitton Y, Cohen-Tannoudji M, Wassef M: Specification of somatosensory area identity in cortical explants. J Neurosci. 1999; 19(12): 4889–98. PubMed Abstract\n\nSato T, Kurihara Y, Asai R, et al.: An endothelin-1 switch specifies maxillomandibular identity. Proc Natl Acad Sci U S A. 2008; 105(48): 18806–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTamarin A, Boyde A: Facial and visceral arch development in the mouse embryo: a study by scanning electron microscopy. J Anat. 1977; 124(Pt 3): 563–80. PubMed Abstract \| Free Full Text\n\nDepew MJ, Liu JK, Long JE, et al.: Dlx5 regulates regional development of the branchial arches and sensory capsules. Development. 1999; 126(17): 3831–46. PubMed Abstract\n\nGitton Y, Benouaiche L, Vincent C, et al.: Dlx5 and Dlx6 expression in the anterior neural fold is essential for patterning the dorsal nasal capsule. Development. 2011; 138(5): 897–903. PubMed Abstract \| Publisher Full Text" }	[ { "id": "3486", "date": "10 Feb 2014", "name": "Jennifer Fish", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, Gitton and colleagues explore the role of Dlx5/6 in upper jaw morphogenesis. Dlx5/6 have largely been recognized for their role in lower jaw identity, based on the fact that loss of these genes in mice results in a loss of lower jaw identity. Previous reports have further suggested that loss of Dlx5/6 in mice causes a transformation of identity from that of lower jaw to upper jaw. In this manuscript, Gitton and colleagues present 3D reconstructions of WT and Dlx5/6 mutant mouse jaws, which allow for a more detailed analysis of the jaw phenotype. They note that the Dlx5/6 jaws not only exhibit dysmorphic lower jaw structures, but the upper jaw elements are also abnormal. They propose two hypotheses that could explain this data: 1) That loss of Dlx5 in the epithelia overlying the developing upper jaw primorida disrupts signaling to the underlying CNC (as previously hypothesized by the Hinge and Caps model of jaw development), or 2) that Dlx5 is transiently expressed in cells that will later populate the maxillary arch, and that this transient expression is essential for subsequent upper jaw morphogenesis. Using lineage tracing experiments, the authors conclude that Dlx5 is indeed transiently expressed in precursors that will populate the maxillary arch, and also provide support for the Hinge and Caps model.The question that Gitton and colleagues proposed is an important one, as the role of Dlx5/6 in jaw morphogenesis is clearly not limited to lower jaw identity. The 3D reconstructions provide improved morphological detail of the Dlx5/6 mutants, and clearly show the abnormal upper jaw morphology in these mutants. The main concern I have with this manuscript as it stands is the way the two hypotheses are described, as well as their interpretation. The first hypothesis refers to Dlx5 expression in the epithelium. It is well known that Dlx5 is expressed in the surface cephalic ectoderm and in the epithelia of the nasal pits, where it is important in regulating the competence of the epithelia to signal to the underlying mesenchyme that gives rise to the nasal capsule and upper jaw. It is this role of Dlx5 in the epithelia that is predicted by, and consistent with, the Hinge and Caps hypothesis. The second hypothesis, as it is phrased, suggests that Dlx5 may be expressed in the mesenchyme of the distal upper jaw. The authors do not say mesenchyme, but this is implied by the phrase \"cells populating the maxillary arch.\" This point needs clarification. If the authors simply mean the epithelium overlying the maxillary arch, this is not really different from hypothesis #1, except to suggest that proliferation of cells near the olfactory pit later contribute to the maxillary epithelium. It does not really provide an alternate biological explanation for the mutant phenotype. Additionally, to clarify this point, it would be nice to see sections of the embryos shown in Figure 2 that would clearly show where Lac-Z is expressed- in the epithelia or the mesenchyme. If it is absent from the mesenchyme, then it is incorrect to say that Dlx5/6 expression (transitory or not) in maxillary arch precursors is essential for upper jaw morphogenesis, as the title suggests.Other minor points:The authors state that CNCCs populating PA1 come from the prosencephalic and anterior mesenchepalic neural folds. In fact, neural crest populating PA1 derives from the posterior mesencephalon and the first and second rhomobomeres of the hindbrain.The authors point out the importance of asymmetric, articulated jaws for predation. It would be more appropriate to say that the evolution of asymmetric jaws has been important for the diversification of vertebrates, as the symmetric jaws of sharks are quite sufficient for predation. This point is also relevant for the evolution of Dlx5/6 expression in the mesenchyme. Although still nested, Dlx gene expression in sharks is distinct from that of mouse and chick, and in fact, Dlx5 expression in shark embryos occurs in the mesenchyme of the upper jaw. This difference in expression may be related to the degree of symmetry in upper and lower jaw morphology (see Compagnucci C et al., 2013).", "responses": [ { "c_id": "802", "date": "06 May 2014", "name": "Giovanni Levi", "role": "Author Response", "response": "We want, first of all, to thank Dr. Fish for her rapid review of our report. Her suggestions gave us the possibility to modify and, in our view, to improve our article taking in account her input. While we thank the reviewer for recognizing the importance of the question addressed in this study and for providing improved morphological analysis showing the abnormal upper jaw morphology of Dlx5/6 mutants, we think that what she calls the “two hypotheses” of this paper needs further consideration.This paper is based on experimental evidence. We are not formulating any hypothesis, but we provide experimental evidence supporting an existing hypothesis: the “Hinge and Caps hypothesis” (for instance Fish JL et al., 2011). We show that indeed cells derived from the frontonasal epithelium after losing the expression of Dlx5/6 migrate to the epithelium overlaying the maxillary arch. This is what we meant saying “cells populating the maxillary arch.”; in no way did we hint to the possibility that mesenchymal cells populating the maxillary arch did express at any time Dlx5/6. The whole text of the manuscript has been reformulated to clarify this point. We have now added a new figure (Figure 3) demonstrating experimentally that derivatives of Dlx5/6 positive cells in the upper jaw are epithelial and not mesenchymal. To make this point even clearer we have changed the title and several sentences of the paper referring now to “Dlx5/6 epithelial precursors”.Regarding the first hypothesis that the reviewer claims that we have formulated: “That loss of Dlx5 in the epithelia overlying the developing upper jaw primorida disrupts signaling to the underlying CNC (as previously hypothesized by the Hinge and Caps model of jaw development)” it is important to note that Dlx5 is NEVER expressed by the epithelia overlying the developing upper jaw primordia. What we show is that derivatives of cells from the frontonasal primordial (FNP) migrate, after having downregulated Dlx5/6, to the upper jaw and then play an important role in defining upper jaw identity. These cells carry therefore a “memory” of having expressed Dlx5/6 before migrating to the epithelia overlying the upper jaw primordia. As the reviewer asks : “, to clarify this point, it would be nice to see sections of the embryos shown in Figure 2 that would clearly show where Lac-Z is expressed- in the epithelia or the mesenchyme.” we have added Figure 3. Other minor points: The authors state that CNCCs populating PA1 come from the prosencephalic and anterior mesenchepalic neural folds. In fact, neural crest populating PA1 derives from the posterior mesencephalon and the first and second rhombomeres of the hindbrain.We removed the sentence as the origin of CNCCs is not particularly relevant to the paper. It would be more appropriate to say that the evolution of asymmetric jaws has been important for the diversification of vertebrates, as the symmetric jaws of sharks are quite sufficient for predation. We agree with the reviewer and the discussion has been modified accordingly including the cited reference.Thanking you again for the time and energy you give to the reviewing process, Sincerely yours, YG, NNN, GL" } ] } ]	1	https://f1000research.com/articles/2-261

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